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What is the main cause of HIV-1 infection in children?
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Mother-to-child transmission (MTCT) is the main cause of HIV-1 infection in children worldwide.
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"BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.",
"The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.",
"Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.",
"High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .",
"DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.",
"To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.",
"The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.",
"Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.",
"Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.",
"The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .",
"These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.",
"Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.",
"Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.",
"First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .",
"1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .",
"Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.",
"Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.",
"The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.",
"Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.",
"Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.",
"Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.",
"Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .",
"H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .",
"The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .",
"Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.",
"We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.",
"A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.",
"Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.",
"To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .",
"In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .",
"The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .",
"As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.",
"Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.",
"H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.",
"Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .",
"Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .",
"To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .",
"Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.",
"It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .",
"Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .",
"Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .",
"Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.",
"However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.",
"Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.",
"Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments."
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What plays the crucial role in the Mother to Child Transmission of HIV-1 and what increases the risk
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DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission.
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"BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.",
"The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.",
"Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.",
"High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .",
"DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.",
"To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.",
"The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.",
"Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.",
"Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.",
"The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .",
"These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.",
"Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.",
"Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.",
"First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .",
"1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .",
"Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.",
"Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.",
"The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.",
"Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.",
"Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.",
"Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.",
"Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .",
"H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .",
"The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .",
"Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.",
"We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.",
"A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.",
"Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.",
"To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .",
"In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .",
"The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .",
"As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.",
"Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.",
"H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.",
"Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .",
"Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .",
"To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .",
"Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.",
"It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .",
"Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .",
"Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .",
"Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.",
"However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.",
"Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.",
"Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments."
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How many children were infected by HIV-1 in 2008-2009, worldwide?
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more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.
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[
"BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.",
"The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.",
"Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.",
"High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .",
"DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.",
"To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.",
"The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.",
"Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.",
"Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.",
"The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .",
"These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.",
"Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.",
"Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.",
"First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .",
"1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .",
"Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.",
"Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.",
"The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.",
"Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.",
"Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.",
"Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.",
"Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .",
"H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .",
"The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .",
"Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.",
"We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.",
"A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.",
"Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.",
"To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .",
"In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .",
"The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .",
"As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.",
"Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.",
"H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.",
"Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .",
"Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .",
"To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .",
"Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.",
"It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .",
"Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .",
"Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .",
"Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.",
"However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.",
"Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.",
"Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments."
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What is the role of C-C Motif Chemokine Ligand 3 Like 1 (CCL3L1) in mother to child transmission of HIV-1?
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High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants
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[
"BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.",
"The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.",
"Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.",
"High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .",
"DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.",
"To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.",
"The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.",
"Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.",
"Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.",
"The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .",
"These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.",
"Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.",
"Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.",
"First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .",
"1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .",
"Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.",
"Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.",
"The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.",
"Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.",
"Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.",
"Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.",
"Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .",
"H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .",
"The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .",
"Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.",
"We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.",
"A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.",
"Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.",
"To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .",
"In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .",
"The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .",
"As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.",
"Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.",
"H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.",
"Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .",
"Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .",
"To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .",
"Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.",
"It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .",
"Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .",
"Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .",
"Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.",
"However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.",
"Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.",
"Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments."
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What is DC-GENR and where is it expressed?
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Dendritic cell-specific ICAM-grabbing non-integrin-related (DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin (L-SIGN)) can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells
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"BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.",
"The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.",
"Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.",
"High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .",
"DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.",
"To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.",
"The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.",
"Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.",
"Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.",
"The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .",
"These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.",
"Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.",
"Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.",
"First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .",
"1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .",
"Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.",
"Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.",
"The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.",
"Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.",
"Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.",
"Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.",
"Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .",
"H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .",
"The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .",
"Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.",
"We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.",
"A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.",
"Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.",
"To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .",
"In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .",
"The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .",
"As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.",
"Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.",
"H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.",
"Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .",
"Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .",
"To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .",
"Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.",
"It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .",
"Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .",
"Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .",
"Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.",
"However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.",
"Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.",
"Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments."
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How does the presence of DC-SIGNR affect the MTCT of HIV-1?
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the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation.
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"BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.",
"The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.",
"Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.",
"High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .",
"DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.",
"To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.",
"The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.",
"Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.",
"Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.",
"The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .",
"These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.",
"Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.",
"Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.",
"First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .",
"1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .",
"Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.",
"Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.",
"The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.",
"Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.",
"Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.",
"Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.",
"Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .",
"H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .",
"The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .",
"Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.",
"We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.",
"A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.",
"Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.",
"To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .",
"In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .",
"The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .",
"As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.",
"Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.",
"H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.",
"Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .",
"Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .",
"To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .",
"Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.",
"It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .",
"Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .",
"Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .",
"Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.",
"However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.",
"Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.",
"Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments."
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Why do low levels of DC-SIGNR enhance Mother to Child Transmission of HIV-1?
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in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1
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"BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.",
"The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.",
"Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.",
"High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .",
"DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.",
"To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.",
"The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.",
"Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.",
"Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.",
"The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .",
"These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.",
"Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.",
"Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.",
"First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .",
"1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .",
"Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.",
"Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.",
"The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.",
"Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.",
"Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.",
"Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.",
"Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .",
"H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .",
"The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .",
"Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.",
"We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.",
"A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.",
"Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.",
"To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .",
"In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .",
"The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .",
"As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.",
"Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.",
"H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.",
"Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .",
"Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .",
"To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .",
"Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.",
"It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .",
"Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .",
"Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .",
"Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.",
"However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.",
"Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.",
"Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments."
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What is the percentage of Mother to Child Transmission of HIV-1, when there is no intervention?
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Without specific interventions, the rate of HIV-1 mother-tochild transmission (MTCT) is approximately 15-45%
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"BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.",
"The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.",
"Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.",
"High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .",
"DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.",
"To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.",
"The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.",
"Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.",
"Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.",
"The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .",
"These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.",
"Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.",
"Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.",
"First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .",
"1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .",
"Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.",
"Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.",
"The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.",
"Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.",
"Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.",
"Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.",
"Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .",
"H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .",
"The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .",
"Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.",
"We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.",
"A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.",
"Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.",
"To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .",
"In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .",
"The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .",
"As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.",
"Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.",
"H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.",
"Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .",
"Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .",
"To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .",
"Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.",
"It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .",
"Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .",
"Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .",
"Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.",
"However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.",
"Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.",
"Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments."
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Does C-C chemokine receptor type 5 (CCR5) affect the transmission of HIV-1?
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Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans
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"BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.",
"The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.",
"Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.",
"High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .",
"DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.",
"To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.",
"The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.",
"Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.",
"Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.",
"The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .",
"These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.",
"Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.",
"Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.",
"First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .",
"1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .",
"Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.",
"Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.",
"The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.",
"Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.",
"Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.",
"Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.",
"Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .",
"H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .",
"The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .",
"Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.",
"We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.",
"A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.",
"Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.",
"To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .",
"In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .",
"The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .",
"As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.",
"Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.",
"H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.",
"Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .",
"Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .",
"To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .",
"Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.",
"It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .",
"Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .",
"Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .",
"Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.",
"However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.",
"Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.",
"Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments."
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How does Mannanose Binding Lectin (MBL) affect elimination of HIV-1 pathogen?
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Mannose-binding lectin (MBL) is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis,
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"BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.",
"The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.",
"Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.",
"High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .",
"DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.",
"To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.",
"The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.",
"Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.",
"Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.",
"The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .",
"These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.",
"Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.",
"Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.",
"First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .",
"1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .",
"Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.",
"Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.",
"The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.",
"Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.",
"Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.",
"Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.",
"Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .",
"H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .",
"The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .",
"Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.",
"We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.",
"A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.",
"Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.",
"To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .",
"In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .",
"The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .",
"As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.",
"Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.",
"H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.",
"Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .",
"Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .",
"To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .",
"Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.",
"It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .",
"Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .",
"Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .",
"Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.",
"However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.",
"Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.",
"Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments."
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How can CCR5's effect in HIV-1 transmission be reduced?
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The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1
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"BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.",
"The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.",
"Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.",
"High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .",
"DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.",
"To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.",
"The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.",
"Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.",
"Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.",
"The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .",
"These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.",
"Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.",
"Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.",
"First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .",
"1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .",
"Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.",
"Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.",
"The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.",
"Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.",
"Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.",
"Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.",
"Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .",
"H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .",
"The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .",
"Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.",
"We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.",
"A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.",
"Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.",
"To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .",
"In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .",
"The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .",
"As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.",
"Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.",
"H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.",
"Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .",
"Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .",
"To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .",
"Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.",
"It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .",
"Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .",
"Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .",
"Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.",
"However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.",
"Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.",
"Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments."
] | 630
| 321
|
What is the size of bovine coronavirus?
|
31 kb
|
[
"We sequenced the first Bovine coronavirus BCoV complete genome sequence from France. This BCoV was directly sequenced from a fecal sample collected from a calf in Normandy in 2014. Text: B ovine coronavirus BCoV belongs to the Nidovirales order, the Coronaviridae family, the Coronavirinae subfamily, and the Betacoronavirus ICTV/proposals/2008.085-122V.v4.Coronaviridae.pdf . Its genome is a single-stranded, linear, and nonsegmented RNA of around 31 kb. BCoV is responsible for respiratory and enteric diseases in cattle, particularly during winter . . To date, the 19 complete BCoV genome sequences available in GenBank databases consulted on 17 January 2017 originated from the United States or Asia.",
". To date, the 19 complete BCoV genome sequences available in GenBank databases consulted on 17 January 2017 originated from the United States or Asia. Here, we report the first complete genome sequence of a BCoV detected in France. The BCoV/FRA-EPI/CAEN/2014/13 strain was obtained from a fecal sample collected from a 1-week-old calf in Normandy in 2014. The presence of BCoV in the fecal sample was assessed using an in-house reverse transcription-PCR RT-PCR targeting the M gene .. A cDNA library was synthesized using SuperScript III Invitrogen, Carlsbad, CA, USA and hexamers. The complete genome sequencing of overlapping PCR products was carried out in both directions, using original primers and Sanger's dideoxy sequencing.",
"The presence of BCoV in the fecal sample was assessed using an in-house reverse transcription-PCR RT-PCR targeting the M gene .. A cDNA library was synthesized using SuperScript III Invitrogen, Carlsbad, CA, USA and hexamers. The complete genome sequencing of overlapping PCR products was carried out in both directions, using original primers and Sanger's dideoxy sequencing. Sequencing reactions were performed as previously described .. Sequences were assembled and annotated using the Geneious software version 5.1.6 . We obtained a sequence counting 30,847 nucleotides. The orf1ab, HE, S, ns5, E, M, and N genes of the obtained BCoV were submitted to a Blastn analysis. According to these analyses, the orf1ab 20kb nucleotides, located at the 5= side of the genome gene is closely related to the Dromedary camel coronavirus DcCoV HKU23-23-362F strain from the United Arab Emirates accession no.",
"The orf1ab, HE, S, ns5, E, M, and N genes of the obtained BCoV were submitted to a Blastn analysis. According to these analyses, the orf1ab 20kb nucleotides, located at the 5= side of the genome gene is closely related to the Dromedary camel coronavirus DcCoV HKU23-23-362F strain from the United Arab Emirates accession no. KF906251 , with a nucleotide identity of 99.19%. Conversely, the NS2, HE, S, ns5, and M genes are closely related to the BCoV Bubalus/Italy/179/07-11 strain accession no. EU019216 , with nucleotide identities of 99.88%, 99.45%, 99.02%, 98.79%, and 99.28%, respectively. The E gene is closely related to the Chinese Bovine coronavirus strain BCV-AKS-01 accession no.",
"EU019216 , with nucleotide identities of 99.88%, 99.45%, 99.02%, 98.79%, and 99.28%, respectively. The E gene is closely related to the Chinese Bovine coronavirus strain BCV-AKS-01 accession no. KU886219 , with a nucleotide identity of 100%. Finally, the highest Blastn score for the N gene was found with the American enteric BCoV-ENT accession no. AF391541 , associated with a nucleotide identity of 100%. Multiple-sequence alignment, including 20 BCoVs and 10 clade A betacoronaviruses closely related to BCoV from North America, two DcCoVs from the United Arab Emirates, and two Human coronavirus OC43 HCoV-OC43 strains from France, was performed using the Muscle algorithm implemented in MEGA7 .",
"AF391541 , associated with a nucleotide identity of 100%. Multiple-sequence alignment, including 20 BCoVs and 10 clade A betacoronaviruses closely related to BCoV from North America, two DcCoVs from the United Arab Emirates, and two Human coronavirus OC43 HCoV-OC43 strains from France, was performed using the Muscle algorithm implemented in MEGA7 . . The phylogenetic analysis on the orf1ab confirms that BCoV/FRA-EPI/CAEN/2014/13 is closely related to the Dromedary camel coronavirus DcCoV HKU23-23-362F. The orf1ab gene of these two viruses together clustered separately from that of BCoV and BCoV-like viruses from North America and Asia. This finding also confirms the results from our previous analysis on partial genomes in which nsp12, S, and N genes of American and Asian BCoVs group together in a cluster tentatively named C 1 .",
"The orf1ab gene of these two viruses together clustered separately from that of BCoV and BCoV-like viruses from North America and Asia. This finding also confirms the results from our previous analysis on partial genomes in which nsp12, S, and N genes of American and Asian BCoVs group together in a cluster tentatively named C 1 . The nsp12 and N coding regions of BCoVs from France and DcCoVs from the United Arab Emirates clustered together in C 2 . The DcCoV S gene individualized from both HCoV-OC43 and BCoV S genes. Potential recombination events could be at the origin of DcCoV. Accession number s .",
"The DcCoV S gene individualized from both HCoV-OC43 and BCoV S genes. Potential recombination events could be at the origin of DcCoV. Accession number s . The complete genome sequence sequence of the BCoV/FRA-EPI/CAEN/2014/13 isolate has been deposited in GenBank under the accession number KX982264."
] | 1,546
| 917
|
What is the molecular structure of bovine coronavirus?
|
single-stranded, linear, and nonsegmented RNA
|
[
"We sequenced the first Bovine coronavirus BCoV complete genome sequence from France. This BCoV was directly sequenced from a fecal sample collected from a calf in Normandy in 2014. Text: B ovine coronavirus BCoV belongs to the Nidovirales order, the Coronaviridae family, the Coronavirinae subfamily, and the Betacoronavirus ICTV/proposals/2008.085-122V.v4.Coronaviridae.pdf . Its genome is a single-stranded, linear, and nonsegmented RNA of around 31 kb. BCoV is responsible for respiratory and enteric diseases in cattle, particularly during winter . . To date, the 19 complete BCoV genome sequences available in GenBank databases consulted on 17 January 2017 originated from the United States or Asia.",
". To date, the 19 complete BCoV genome sequences available in GenBank databases consulted on 17 January 2017 originated from the United States or Asia. Here, we report the first complete genome sequence of a BCoV detected in France. The BCoV/FRA-EPI/CAEN/2014/13 strain was obtained from a fecal sample collected from a 1-week-old calf in Normandy in 2014. The presence of BCoV in the fecal sample was assessed using an in-house reverse transcription-PCR RT-PCR targeting the M gene .. A cDNA library was synthesized using SuperScript III Invitrogen, Carlsbad, CA, USA and hexamers. The complete genome sequencing of overlapping PCR products was carried out in both directions, using original primers and Sanger's dideoxy sequencing.",
"The presence of BCoV in the fecal sample was assessed using an in-house reverse transcription-PCR RT-PCR targeting the M gene .. A cDNA library was synthesized using SuperScript III Invitrogen, Carlsbad, CA, USA and hexamers. The complete genome sequencing of overlapping PCR products was carried out in both directions, using original primers and Sanger's dideoxy sequencing. Sequencing reactions were performed as previously described .. Sequences were assembled and annotated using the Geneious software version 5.1.6 . We obtained a sequence counting 30,847 nucleotides. The orf1ab, HE, S, ns5, E, M, and N genes of the obtained BCoV were submitted to a Blastn analysis. According to these analyses, the orf1ab 20kb nucleotides, located at the 5= side of the genome gene is closely related to the Dromedary camel coronavirus DcCoV HKU23-23-362F strain from the United Arab Emirates accession no.",
"The orf1ab, HE, S, ns5, E, M, and N genes of the obtained BCoV were submitted to a Blastn analysis. According to these analyses, the orf1ab 20kb nucleotides, located at the 5= side of the genome gene is closely related to the Dromedary camel coronavirus DcCoV HKU23-23-362F strain from the United Arab Emirates accession no. KF906251 , with a nucleotide identity of 99.19%. Conversely, the NS2, HE, S, ns5, and M genes are closely related to the BCoV Bubalus/Italy/179/07-11 strain accession no. EU019216 , with nucleotide identities of 99.88%, 99.45%, 99.02%, 98.79%, and 99.28%, respectively. The E gene is closely related to the Chinese Bovine coronavirus strain BCV-AKS-01 accession no.",
"EU019216 , with nucleotide identities of 99.88%, 99.45%, 99.02%, 98.79%, and 99.28%, respectively. The E gene is closely related to the Chinese Bovine coronavirus strain BCV-AKS-01 accession no. KU886219 , with a nucleotide identity of 100%. Finally, the highest Blastn score for the N gene was found with the American enteric BCoV-ENT accession no. AF391541 , associated with a nucleotide identity of 100%. Multiple-sequence alignment, including 20 BCoVs and 10 clade A betacoronaviruses closely related to BCoV from North America, two DcCoVs from the United Arab Emirates, and two Human coronavirus OC43 HCoV-OC43 strains from France, was performed using the Muscle algorithm implemented in MEGA7 .",
"AF391541 , associated with a nucleotide identity of 100%. Multiple-sequence alignment, including 20 BCoVs and 10 clade A betacoronaviruses closely related to BCoV from North America, two DcCoVs from the United Arab Emirates, and two Human coronavirus OC43 HCoV-OC43 strains from France, was performed using the Muscle algorithm implemented in MEGA7 . . The phylogenetic analysis on the orf1ab confirms that BCoV/FRA-EPI/CAEN/2014/13 is closely related to the Dromedary camel coronavirus DcCoV HKU23-23-362F. The orf1ab gene of these two viruses together clustered separately from that of BCoV and BCoV-like viruses from North America and Asia. This finding also confirms the results from our previous analysis on partial genomes in which nsp12, S, and N genes of American and Asian BCoVs group together in a cluster tentatively named C 1 .",
"The orf1ab gene of these two viruses together clustered separately from that of BCoV and BCoV-like viruses from North America and Asia. This finding also confirms the results from our previous analysis on partial genomes in which nsp12, S, and N genes of American and Asian BCoVs group together in a cluster tentatively named C 1 . The nsp12 and N coding regions of BCoVs from France and DcCoVs from the United Arab Emirates clustered together in C 2 . The DcCoV S gene individualized from both HCoV-OC43 and BCoV S genes. Potential recombination events could be at the origin of DcCoV. Accession number s .",
"The DcCoV S gene individualized from both HCoV-OC43 and BCoV S genes. Potential recombination events could be at the origin of DcCoV. Accession number s . The complete genome sequence sequence of the BCoV/FRA-EPI/CAEN/2014/13 isolate has been deposited in GenBank under the accession number KX982264."
] | 1,546
| 918
|
How many nucleotides does bovine coronavirus contain?
|
30,847 nucleotides
|
[
"We sequenced the first Bovine coronavirus BCoV complete genome sequence from France. This BCoV was directly sequenced from a fecal sample collected from a calf in Normandy in 2014. Text: B ovine coronavirus BCoV belongs to the Nidovirales order, the Coronaviridae family, the Coronavirinae subfamily, and the Betacoronavirus ICTV/proposals/2008.085-122V.v4.Coronaviridae.pdf . Its genome is a single-stranded, linear, and nonsegmented RNA of around 31 kb. BCoV is responsible for respiratory and enteric diseases in cattle, particularly during winter . . To date, the 19 complete BCoV genome sequences available in GenBank databases consulted on 17 January 2017 originated from the United States or Asia.",
". To date, the 19 complete BCoV genome sequences available in GenBank databases consulted on 17 January 2017 originated from the United States or Asia. Here, we report the first complete genome sequence of a BCoV detected in France. The BCoV/FRA-EPI/CAEN/2014/13 strain was obtained from a fecal sample collected from a 1-week-old calf in Normandy in 2014. The presence of BCoV in the fecal sample was assessed using an in-house reverse transcription-PCR RT-PCR targeting the M gene .. A cDNA library was synthesized using SuperScript III Invitrogen, Carlsbad, CA, USA and hexamers. The complete genome sequencing of overlapping PCR products was carried out in both directions, using original primers and Sanger's dideoxy sequencing.",
"The presence of BCoV in the fecal sample was assessed using an in-house reverse transcription-PCR RT-PCR targeting the M gene .. A cDNA library was synthesized using SuperScript III Invitrogen, Carlsbad, CA, USA and hexamers. The complete genome sequencing of overlapping PCR products was carried out in both directions, using original primers and Sanger's dideoxy sequencing. Sequencing reactions were performed as previously described .. Sequences were assembled and annotated using the Geneious software version 5.1.6 . We obtained a sequence counting 30,847 nucleotides. The orf1ab, HE, S, ns5, E, M, and N genes of the obtained BCoV were submitted to a Blastn analysis. According to these analyses, the orf1ab 20kb nucleotides, located at the 5= side of the genome gene is closely related to the Dromedary camel coronavirus DcCoV HKU23-23-362F strain from the United Arab Emirates accession no.",
"The orf1ab, HE, S, ns5, E, M, and N genes of the obtained BCoV were submitted to a Blastn analysis. According to these analyses, the orf1ab 20kb nucleotides, located at the 5= side of the genome gene is closely related to the Dromedary camel coronavirus DcCoV HKU23-23-362F strain from the United Arab Emirates accession no. KF906251 , with a nucleotide identity of 99.19%. Conversely, the NS2, HE, S, ns5, and M genes are closely related to the BCoV Bubalus/Italy/179/07-11 strain accession no. EU019216 , with nucleotide identities of 99.88%, 99.45%, 99.02%, 98.79%, and 99.28%, respectively. The E gene is closely related to the Chinese Bovine coronavirus strain BCV-AKS-01 accession no.",
"EU019216 , with nucleotide identities of 99.88%, 99.45%, 99.02%, 98.79%, and 99.28%, respectively. The E gene is closely related to the Chinese Bovine coronavirus strain BCV-AKS-01 accession no. KU886219 , with a nucleotide identity of 100%. Finally, the highest Blastn score for the N gene was found with the American enteric BCoV-ENT accession no. AF391541 , associated with a nucleotide identity of 100%. Multiple-sequence alignment, including 20 BCoVs and 10 clade A betacoronaviruses closely related to BCoV from North America, two DcCoVs from the United Arab Emirates, and two Human coronavirus OC43 HCoV-OC43 strains from France, was performed using the Muscle algorithm implemented in MEGA7 .",
"AF391541 , associated with a nucleotide identity of 100%. Multiple-sequence alignment, including 20 BCoVs and 10 clade A betacoronaviruses closely related to BCoV from North America, two DcCoVs from the United Arab Emirates, and two Human coronavirus OC43 HCoV-OC43 strains from France, was performed using the Muscle algorithm implemented in MEGA7 . . The phylogenetic analysis on the orf1ab confirms that BCoV/FRA-EPI/CAEN/2014/13 is closely related to the Dromedary camel coronavirus DcCoV HKU23-23-362F. The orf1ab gene of these two viruses together clustered separately from that of BCoV and BCoV-like viruses from North America and Asia. This finding also confirms the results from our previous analysis on partial genomes in which nsp12, S, and N genes of American and Asian BCoVs group together in a cluster tentatively named C 1 .",
"The orf1ab gene of these two viruses together clustered separately from that of BCoV and BCoV-like viruses from North America and Asia. This finding also confirms the results from our previous analysis on partial genomes in which nsp12, S, and N genes of American and Asian BCoVs group together in a cluster tentatively named C 1 . The nsp12 and N coding regions of BCoVs from France and DcCoVs from the United Arab Emirates clustered together in C 2 . The DcCoV S gene individualized from both HCoV-OC43 and BCoV S genes. Potential recombination events could be at the origin of DcCoV. Accession number s .",
"The DcCoV S gene individualized from both HCoV-OC43 and BCoV S genes. Potential recombination events could be at the origin of DcCoV. Accession number s . The complete genome sequence sequence of the BCoV/FRA-EPI/CAEN/2014/13 isolate has been deposited in GenBank under the accession number KX982264."
] | 1,546
| 919
|
What is the size of the orf1ab gene in bovine coronavirus?
|
20kb
|
[
"We sequenced the first Bovine coronavirus BCoV complete genome sequence from France. This BCoV was directly sequenced from a fecal sample collected from a calf in Normandy in 2014. Text: B ovine coronavirus BCoV belongs to the Nidovirales order, the Coronaviridae family, the Coronavirinae subfamily, and the Betacoronavirus ICTV/proposals/2008.085-122V.v4.Coronaviridae.pdf . Its genome is a single-stranded, linear, and nonsegmented RNA of around 31 kb. BCoV is responsible for respiratory and enteric diseases in cattle, particularly during winter . . To date, the 19 complete BCoV genome sequences available in GenBank databases consulted on 17 January 2017 originated from the United States or Asia.",
". To date, the 19 complete BCoV genome sequences available in GenBank databases consulted on 17 January 2017 originated from the United States or Asia. Here, we report the first complete genome sequence of a BCoV detected in France. The BCoV/FRA-EPI/CAEN/2014/13 strain was obtained from a fecal sample collected from a 1-week-old calf in Normandy in 2014. The presence of BCoV in the fecal sample was assessed using an in-house reverse transcription-PCR RT-PCR targeting the M gene .. A cDNA library was synthesized using SuperScript III Invitrogen, Carlsbad, CA, USA and hexamers. The complete genome sequencing of overlapping PCR products was carried out in both directions, using original primers and Sanger's dideoxy sequencing.",
"The presence of BCoV in the fecal sample was assessed using an in-house reverse transcription-PCR RT-PCR targeting the M gene .. A cDNA library was synthesized using SuperScript III Invitrogen, Carlsbad, CA, USA and hexamers. The complete genome sequencing of overlapping PCR products was carried out in both directions, using original primers and Sanger's dideoxy sequencing. Sequencing reactions were performed as previously described .. Sequences were assembled and annotated using the Geneious software version 5.1.6 . We obtained a sequence counting 30,847 nucleotides. The orf1ab, HE, S, ns5, E, M, and N genes of the obtained BCoV were submitted to a Blastn analysis. According to these analyses, the orf1ab 20kb nucleotides, located at the 5= side of the genome gene is closely related to the Dromedary camel coronavirus DcCoV HKU23-23-362F strain from the United Arab Emirates accession no.",
"The orf1ab, HE, S, ns5, E, M, and N genes of the obtained BCoV were submitted to a Blastn analysis. According to these analyses, the orf1ab 20kb nucleotides, located at the 5= side of the genome gene is closely related to the Dromedary camel coronavirus DcCoV HKU23-23-362F strain from the United Arab Emirates accession no. KF906251 , with a nucleotide identity of 99.19%. Conversely, the NS2, HE, S, ns5, and M genes are closely related to the BCoV Bubalus/Italy/179/07-11 strain accession no. EU019216 , with nucleotide identities of 99.88%, 99.45%, 99.02%, 98.79%, and 99.28%, respectively. The E gene is closely related to the Chinese Bovine coronavirus strain BCV-AKS-01 accession no.",
"EU019216 , with nucleotide identities of 99.88%, 99.45%, 99.02%, 98.79%, and 99.28%, respectively. The E gene is closely related to the Chinese Bovine coronavirus strain BCV-AKS-01 accession no. KU886219 , with a nucleotide identity of 100%. Finally, the highest Blastn score for the N gene was found with the American enteric BCoV-ENT accession no. AF391541 , associated with a nucleotide identity of 100%. Multiple-sequence alignment, including 20 BCoVs and 10 clade A betacoronaviruses closely related to BCoV from North America, two DcCoVs from the United Arab Emirates, and two Human coronavirus OC43 HCoV-OC43 strains from France, was performed using the Muscle algorithm implemented in MEGA7 .",
"AF391541 , associated with a nucleotide identity of 100%. Multiple-sequence alignment, including 20 BCoVs and 10 clade A betacoronaviruses closely related to BCoV from North America, two DcCoVs from the United Arab Emirates, and two Human coronavirus OC43 HCoV-OC43 strains from France, was performed using the Muscle algorithm implemented in MEGA7 . . The phylogenetic analysis on the orf1ab confirms that BCoV/FRA-EPI/CAEN/2014/13 is closely related to the Dromedary camel coronavirus DcCoV HKU23-23-362F. The orf1ab gene of these two viruses together clustered separately from that of BCoV and BCoV-like viruses from North America and Asia. This finding also confirms the results from our previous analysis on partial genomes in which nsp12, S, and N genes of American and Asian BCoVs group together in a cluster tentatively named C 1 .",
"The orf1ab gene of these two viruses together clustered separately from that of BCoV and BCoV-like viruses from North America and Asia. This finding also confirms the results from our previous analysis on partial genomes in which nsp12, S, and N genes of American and Asian BCoVs group together in a cluster tentatively named C 1 . The nsp12 and N coding regions of BCoVs from France and DcCoVs from the United Arab Emirates clustered together in C 2 . The DcCoV S gene individualized from both HCoV-OC43 and BCoV S genes. Potential recombination events could be at the origin of DcCoV. Accession number s .",
"The DcCoV S gene individualized from both HCoV-OC43 and BCoV S genes. Potential recombination events could be at the origin of DcCoV. Accession number s . The complete genome sequence sequence of the BCoV/FRA-EPI/CAEN/2014/13 isolate has been deposited in GenBank under the accession number KX982264."
] | 1,546
| 920
|
Is the orf1ab gene at the 3' or 5' end of the bovine coronavirus genome?
|
5= side
|
[
"We sequenced the first Bovine coronavirus BCoV complete genome sequence from France. This BCoV was directly sequenced from a fecal sample collected from a calf in Normandy in 2014. Text: B ovine coronavirus BCoV belongs to the Nidovirales order, the Coronaviridae family, the Coronavirinae subfamily, and the Betacoronavirus ICTV/proposals/2008.085-122V.v4.Coronaviridae.pdf . Its genome is a single-stranded, linear, and nonsegmented RNA of around 31 kb. BCoV is responsible for respiratory and enteric diseases in cattle, particularly during winter . . To date, the 19 complete BCoV genome sequences available in GenBank databases consulted on 17 January 2017 originated from the United States or Asia.",
". To date, the 19 complete BCoV genome sequences available in GenBank databases consulted on 17 January 2017 originated from the United States or Asia. Here, we report the first complete genome sequence of a BCoV detected in France. The BCoV/FRA-EPI/CAEN/2014/13 strain was obtained from a fecal sample collected from a 1-week-old calf in Normandy in 2014. The presence of BCoV in the fecal sample was assessed using an in-house reverse transcription-PCR RT-PCR targeting the M gene .. A cDNA library was synthesized using SuperScript III Invitrogen, Carlsbad, CA, USA and hexamers. The complete genome sequencing of overlapping PCR products was carried out in both directions, using original primers and Sanger's dideoxy sequencing.",
"The presence of BCoV in the fecal sample was assessed using an in-house reverse transcription-PCR RT-PCR targeting the M gene .. A cDNA library was synthesized using SuperScript III Invitrogen, Carlsbad, CA, USA and hexamers. The complete genome sequencing of overlapping PCR products was carried out in both directions, using original primers and Sanger's dideoxy sequencing. Sequencing reactions were performed as previously described .. Sequences were assembled and annotated using the Geneious software version 5.1.6 . We obtained a sequence counting 30,847 nucleotides. The orf1ab, HE, S, ns5, E, M, and N genes of the obtained BCoV were submitted to a Blastn analysis. According to these analyses, the orf1ab 20kb nucleotides, located at the 5= side of the genome gene is closely related to the Dromedary camel coronavirus DcCoV HKU23-23-362F strain from the United Arab Emirates accession no.",
"The orf1ab, HE, S, ns5, E, M, and N genes of the obtained BCoV were submitted to a Blastn analysis. According to these analyses, the orf1ab 20kb nucleotides, located at the 5= side of the genome gene is closely related to the Dromedary camel coronavirus DcCoV HKU23-23-362F strain from the United Arab Emirates accession no. KF906251 , with a nucleotide identity of 99.19%. Conversely, the NS2, HE, S, ns5, and M genes are closely related to the BCoV Bubalus/Italy/179/07-11 strain accession no. EU019216 , with nucleotide identities of 99.88%, 99.45%, 99.02%, 98.79%, and 99.28%, respectively. The E gene is closely related to the Chinese Bovine coronavirus strain BCV-AKS-01 accession no.",
"EU019216 , with nucleotide identities of 99.88%, 99.45%, 99.02%, 98.79%, and 99.28%, respectively. The E gene is closely related to the Chinese Bovine coronavirus strain BCV-AKS-01 accession no. KU886219 , with a nucleotide identity of 100%. Finally, the highest Blastn score for the N gene was found with the American enteric BCoV-ENT accession no. AF391541 , associated with a nucleotide identity of 100%. Multiple-sequence alignment, including 20 BCoVs and 10 clade A betacoronaviruses closely related to BCoV from North America, two DcCoVs from the United Arab Emirates, and two Human coronavirus OC43 HCoV-OC43 strains from France, was performed using the Muscle algorithm implemented in MEGA7 .",
"AF391541 , associated with a nucleotide identity of 100%. Multiple-sequence alignment, including 20 BCoVs and 10 clade A betacoronaviruses closely related to BCoV from North America, two DcCoVs from the United Arab Emirates, and two Human coronavirus OC43 HCoV-OC43 strains from France, was performed using the Muscle algorithm implemented in MEGA7 . . The phylogenetic analysis on the orf1ab confirms that BCoV/FRA-EPI/CAEN/2014/13 is closely related to the Dromedary camel coronavirus DcCoV HKU23-23-362F. The orf1ab gene of these two viruses together clustered separately from that of BCoV and BCoV-like viruses from North America and Asia. This finding also confirms the results from our previous analysis on partial genomes in which nsp12, S, and N genes of American and Asian BCoVs group together in a cluster tentatively named C 1 .",
"The orf1ab gene of these two viruses together clustered separately from that of BCoV and BCoV-like viruses from North America and Asia. This finding also confirms the results from our previous analysis on partial genomes in which nsp12, S, and N genes of American and Asian BCoVs group together in a cluster tentatively named C 1 . The nsp12 and N coding regions of BCoVs from France and DcCoVs from the United Arab Emirates clustered together in C 2 . The DcCoV S gene individualized from both HCoV-OC43 and BCoV S genes. Potential recombination events could be at the origin of DcCoV. Accession number s .",
"The DcCoV S gene individualized from both HCoV-OC43 and BCoV S genes. Potential recombination events could be at the origin of DcCoV. Accession number s . The complete genome sequence sequence of the BCoV/FRA-EPI/CAEN/2014/13 isolate has been deposited in GenBank under the accession number KX982264."
] | 1,546
| 921
|
What is a significant cause of Influenze like illness among healthy adolescents and adults presenting for medical evaluation?
|
HCoV
|
[
"Human coronavirus HCoV is a known cause of influenza‐like illness ILI . In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 34% , HCoV‐229E 28% , HCoV‐NL63 22% , and HCoV‐HKU1 16% . We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%.",
"Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%. By contrast, other HCoV species HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E are much more prevalent, much less severe, and common causes of influenza-like illness ILI . Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection.",
"6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded.",
"Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 .",
"Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus HCoV is an important cause of influenza-like illness ILI ranged from 4% to 22%. 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known.",
"8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation.",
"Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease."
] | 1,545
| 1,658
|
What is the most common species of Human Coronavirus among adults?
|
HCoV-OC43
|
[
"Human coronavirus HCoV is a known cause of influenza‐like illness ILI . In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 34% , HCoV‐229E 28% , HCoV‐NL63 22% , and HCoV‐HKU1 16% . We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%.",
"Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%. By contrast, other HCoV species HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E are much more prevalent, much less severe, and common causes of influenza-like illness ILI . Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection.",
"6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded.",
"Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 .",
"Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus HCoV is an important cause of influenza-like illness ILI ranged from 4% to 22%. 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known.",
"8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation.",
"Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease."
] | 1,545
| 1,659
|
Which Human Coronavirus showed species specific clinical characteristics of its infection?
|
HCoV‐HKU1
|
[
"Human coronavirus HCoV is a known cause of influenza‐like illness ILI . In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 34% , HCoV‐229E 28% , HCoV‐NL63 22% , and HCoV‐HKU1 16% . We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%.",
"Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%. By contrast, other HCoV species HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E are much more prevalent, much less severe, and common causes of influenza-like illness ILI . Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection.",
"6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded.",
"Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 .",
"Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus HCoV is an important cause of influenza-like illness ILI ranged from 4% to 22%. 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known.",
"8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation.",
"Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease."
] | 1,545
| 1,660
|
What causes the outbreak of SARS and MERS.
|
Highly virulent species of HCoV
|
[
"Human coronavirus HCoV is a known cause of influenza‐like illness ILI . In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 34% , HCoV‐229E 28% , HCoV‐NL63 22% , and HCoV‐HKU1 16% . We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%.",
"Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%. By contrast, other HCoV species HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E are much more prevalent, much less severe, and common causes of influenza-like illness ILI . Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection.",
"6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded.",
"Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 .",
"Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus HCoV is an important cause of influenza-like illness ILI ranged from 4% to 22%. 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known.",
"8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation.",
"Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease."
] | 1,545
| 1,717
|
What is the case fatality rate of SARS and MERS?
|
ranged from 14% to 45%
|
[
"Human coronavirus HCoV is a known cause of influenza‐like illness ILI . In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 34% , HCoV‐229E 28% , HCoV‐NL63 22% , and HCoV‐HKU1 16% . We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%.",
"Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%. By contrast, other HCoV species HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E are much more prevalent, much less severe, and common causes of influenza-like illness ILI . Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection.",
"6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded.",
"Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 .",
"Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus HCoV is an important cause of influenza-like illness ILI ranged from 4% to 22%. 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known.",
"8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation.",
"Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease."
] | 1,545
| 1,718
|
What were the common HCOV strains in the 5 year USA study?
|
HCoV-OC43 and HCoV-229E
|
[
"Human coronavirus HCoV is a known cause of influenza‐like illness ILI . In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 34% , HCoV‐229E 28% , HCoV‐NL63 22% , and HCoV‐HKU1 16% . We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%.",
"Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%. By contrast, other HCoV species HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E are much more prevalent, much less severe, and common causes of influenza-like illness ILI . Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection.",
"6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded.",
"Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 .",
"Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus HCoV is an important cause of influenza-like illness ILI ranged from 4% to 22%. 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known.",
"8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation.",
"Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease."
] | 1,545
| 1,719
|
Which species are more prevalent but less severe?
|
HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E
|
[
"Human coronavirus HCoV is a known cause of influenza‐like illness ILI . In a multisite, observational, longitudinal study of ILI among otherwise healthy adolescents and adults, 12% of subjects were PCR‐positive for HCoV. The distribution of species was as follows: HCoV‐OC43 34% , HCoV‐229E 28% , HCoV‐NL63 22% , and HCoV‐HKU1 16% . We did not observe species‐specific differences in the clinical characteristics of HCoV infection, with the exception of HCoV‐HKU1, for which the severity of gastrointestinal symptoms trended higher on the fourth day of illness. Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%.",
"Text: Clinical manifestations of human coronavirus HCoV infection range from a mild, self-limiting illness of the upper respiratory tract to an acute respiratory distress syndrome with a high mortality rate. Highly virulent species of HCoV were responsible for outbreaks of severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS ; case-fatality rates ranged from 14% to 45%. By contrast, other HCoV species HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E are much more prevalent, much less severe, and common causes of influenza-like illness ILI . Five previous studies have described the species-specific clinical characteristics of HCoV infection among adults. 6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection.",
"6, 7, In two of these studies, a significant proportion of the study population had underlying medical conditions. 6, 7 Herein, we describe, among a cohort of otherwise healthy adolescents and adults with influenza-like illness ILI , the species-specific prevalence and severity of symptoms associated with HCoV infection. 13 Patients 0-65 years of age and presenting for care <72 hours after onset of ILI symptoms were recruited for study participation. ILI was defined as a temperature ≥100.4°F and sore throat or one of the following respiratory symptoms: cough, sputum production, shortness of breath, or chest pain. Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded.",
"Both inpatient and outpatient subjects were eligible to participate. Patients with underlying medical conditions eg, diabetes, chronic obstructive pulmonary disease, severe asthma , women with a high-risk or complicated pregnancy, and patients with a poorly controlled psychiatric disorder were excluded. Information on patient demographics and presence/severity of symptoms at the time of enrollment was collected by in-person interview. Participants were then instructed on the use of a daily diary to record the presence/severity of symptoms for 7 days following initial symptom onset. Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 .",
"Symptom severity was rated on an ordinal scale from 0 none to 3 severe . Symptom severity scores were quantified using the following five measures: i individual symptom score for 20 symptoms, ii the upper respiratory symptom score, calculated as the sum of severity scores for earache, runny nose, sore throat, and sneezing, iii the lower respiratory symptom score, calculated as the sum of severity scores for cough, difficulty breathing, hoarseness, and chest discomfort, iv the gastrointestinal symptom score, calculated as the sum of severity scores for diarrhea, vomiting, anorexia, nausea, and Table 1 . There was season-to-season variability in the leading causes of The findings of our study, conducted over a 5-year period at five geographically dispersed sites in the USA, demonstrate that human coronavirus HCoV is an important cause of influenza-like illness ILI ranged from 4% to 22%. 14 Additionally, we found HCoV-OC43 to be the most common species among adults, as has been reported elsewhere. 8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known.",
"8, 9, 11, 12, 14 HCoV-OC43 and HCoV-229E were the most common strains in alternate seasons, reflecting a season-to-season variability of HCoV strain circulation that has been reported in other multiyear studies. 4 8 The mechanisms by which this particular species elicits these symptoms are not known. The strengths of this study of HCoV in otherwise healthy adolescents and adults include its multisite and multiyear design, the use of a multiplex diagnostic panel, the prospective collection of symptom data, and the use of a symptom severity scale similar to what has been employed previously. 15 One important limitation of this study was our selective recruitment of individuals who had presented to a healthcare facility for care of an ILI. Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation.",
"Therefore, our cases are not representative of HCoV infection in the community, where individuals with mild, self-limiting illness due to HCoV opt not to seek medical care for the management of their ILI. In summary, we have shown that HCoV is a significant cause of ILI among otherwise healthy adolescents and adults presenting for medical evaluation. Although there were differences in species distribution by age group, we did not detect any differences between species with respect to the clinical spectrum of disease."
] | 1,545
| 1,720
|
What kinds of viruses are Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV)?
|
arboviruses
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553
| 3,003
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What are the current clinically-available methods to detect encephalitis viral antigens?
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ELISA and IFA
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553
| 3,004
|
What methods exist for detecting multiple antigens simultaneously in a one-sample, laboratory test?
|
two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553
| 3,005
|
How many antigens could be detected by Liew's multiplex ELISA test?
|
9
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
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What kind of antibodies were used in the ELISA-array assay?
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monoclonal
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"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553
| 3,007
|
How was the ELISA assay validated?
|
using cultured viruses and inoculated chicken eggs with patient sera
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553
| 3,008
|
What capture antibodies were used in the study?
|
4D5, 2B5, 1F1, 2B8, 4F9, and 4E11
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553
| 3,009
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What was the spotting concentration range for the capture antibodies?
|
from 0.2 to 0.0125 mg/ml
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553
| 3,010
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How was the proper spotting concentration determined?
|
combination of minimized cross reaction and higher signal intensity
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
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How was cross reaction detection determined?
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by applying JEV, YF, and DV cultures
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[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553
| 3,012
|
How was the ELISA-array assay validated?
|
using cultured viruses and inoculated chicken eggs with patient sera
|
[
"Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , and eastern equine encephalitis virus EEEV can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array.",
"An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a \"sandwich\" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.",
"This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use. Text: Japanese encephalitis virus JEV , tick-borne encephalitis virus TBEV , eastern equine encephalitis virus EEEV , sindbis virus SV , and dengue virus DV are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology .",
"Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay . There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods . Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported.",
"Liew validated one multiplex ELISA for the detection of 9 antigens; Anderson used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus.",
"The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array multiplex and ease of use and has potential for the identification of clinical encephalitis virus. Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose . Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures.",
"Specific monoclonal antibodies 4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus were selected for this study. All of the antibodies were raised according to standard procedures. Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies 2A10, 1 F1, and 4E11 were coupled to biotin-NHS ester Pierce, Germany at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column Pierce . Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.",
"Immunologic reactions were reported by Streptavidin-HRP CWBIO, Beijing, China and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control. JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.",
"Viral titers were determined by the 50% tissue culture infectious dose TCID 50 method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone. Antibodies were spotted using a BIODOT machine BD6000;California, USA on ELISA plates 30 nl/dot . The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay.",
"Then, the plates were dried, sealed, and stored at 4°C before use . When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline PBS , PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.",
"A range of monoclonal antibody concentrations 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml were compared. Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation. Antigen binding was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h, followed by washing 3 times 1 × PBS containing 0.1% Tween-20 . Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS containing 0.1% Tween-20 and 5% FCS at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate Thermo scientific, Rockford, USA . Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software.",
"Visualization of the plate was performed in AE 1000 cool CCD image analyzer Beijing BGI GBI Biotech Company., LTD, China . The signal intensity and background of each spot was read out and recorded with \"Monster\"software. The positive signals were defined as a signal value > 400 and a signal value sample /signal value negative > 2. The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate .",
"The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS pH 7.4 , and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate . Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use.",
"3 days later, the liquid was collected and divided into two portions one for inactivation and one for RNA extraction . The RNA and inactivated samples were stored at -70°C before use. RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit Qiagen Inc., Valencia, CA, USA according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes .",
"The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit Qiagen Inc, . The reaction consisted of 10 μL of 2 × reaction buffer 0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes . RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 Roche, Switzerland . Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold .",
"Optimization of the ELISA-array assay The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison. The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml each was serially diluted 2-fold . The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full .",
"Cross reaction detection was also conducted by applying JEV, YF, and DV cultures. Spot morphology observation Figures 2a, b , and 2c demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology round and full . Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d .",
"Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear Figure 2d . Consequently, spotting concentration optimization of other capture antibodies 4D5, 1F1, 4E11, and 2B8 demonstrated that 0.05 mg/ml was also suitable data not shown . The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments. Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined.",
"Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared Table 1 . The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.",
"The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus YFV cultures 5 × 10 5 TCID 50 /ml, West Nile virus WNV cultures 2 × 10 6 TCID 50 /ml , and Western equine encephalitis virus 1 × 10 7 TCID 50 /ml . The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity. Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody 2A10, 4E11, and 1F1 was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay.",
"The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results Figures 4 and 5 . Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV Figure 6b . The ELISA-array assay suggested that the three patients were all infected with TBEV. To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable.",
"The results were also positive Figure 6a . The consensus detection results confirmed that the ELISAarray assay was reliable. To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.",
"Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements. It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity.",
"The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies.",
"The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same 1F1 and 4E11, respectively , the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.",
"The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use.",
"Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods heating and β-propiolactone were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method.",
"Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates.",
"The time required for the ELISA-array is significantly less than for conventional ELISA 4 h vs. a minimum of 6 h, respectively . Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use."
] | 1,553
| 3,013
|
In 2010, how many cases of tuberculosis were estimated in China?
|
108 per 100,000
|
[
"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
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What is the population of Shandong province?
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94 million
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[
"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
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What was the purpose of this study?
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estimate the TB prevalence in Shandong
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"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
] | 1,557
| 3,016
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What was the age range for the people surveyed?
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15 years old or above
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[
"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
] | 1,557
| 3,017
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How was the survey designed?
|
in accordance with WHO recommendations
|
[
"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
] | 1,557
| 3,018
|
Was was the sample size?
|
52500
|
[
"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
] | 1,557
| 3,019
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How were the clusters selected?
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A stratified multi stage random sampling
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[
"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
] | 1,557
| 3,020
|
How many people were in a community cluster?
|
1250 to 1750
|
[
"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
] | 1,557
| 3,021
|
Who was excluded from the study?
|
Military barracks and prisons
|
[
"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
] | 1,557
| 3,022
|
When was the study conducted?
|
March to June 2010
|
[
"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
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Who conducted the study?
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clinicians, public health doctors, radiologists, laboratory technicians and nurses
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[
"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
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What medium was used to collect the sputum samples?
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Löwenstein-Jensen medium
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"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
] | 1,557
| 3,025
|
What was the response rate for the study?
|
95% to 97%
|
[
"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
] | 1,557
| 3,026
|
What was the average age of a study participant?
|
46 years
|
[
"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
] | 1,557
| 3,027
|
What was the prevalence rate in Shandong in 2010 for sputum positive cases of tuberculosis?
|
22.1
|
[
"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
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What was the most striking finding of the study regarding tuberculosis patients?
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a large proportion of TB patients did not present consistent cough
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"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
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How many cases of sputum positive tuberculosis patients had no persistent cough?
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"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
] | 1,557
| 3,030
|
How many tuberculosis patients in Shandong were over 65 years old?
|
over half
|
[
"BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.",
"Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.",
"The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .",
"Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .",
"Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.",
"The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .",
"The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .",
"The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.",
"Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.",
"While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.",
"All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.",
"Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.",
"A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.",
"Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.",
"CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .",
"The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.",
"Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.",
"Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.",
"The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .",
"Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.",
"The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .",
"Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .",
"Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.",
"Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.",
"The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.",
"Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .",
"CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.",
"The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .",
"Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .",
"Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.",
"Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.",
"As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.",
"How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .",
"For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.",
"However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.",
"Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests."
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How many surgical masks or respirators have past studies projected will be required for a pandemic in the United States?
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an estimated 7.3 billion
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"Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323.:1129-1130. .1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview 25:53 COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A H1N1 virus H1N1 pdm09 pandemic or the severe acute respiratory syndrome coronavirus SARS-CoV or the Middle East respiratory syndrome coronavirus MERS-CoV epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness.",
"Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A H1N1 pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe.",
"By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe. SARS-CoV . and MERS-CoV 2012-current cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV indicating sustained and even worldwide transmission could occur , and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals.",
"SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries.",
"Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical border and school closing, infection control measures and pharmaceutical antiviral prophylaxis, vaccines interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance.",
"Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention CDC distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States.",
"Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms.",
"However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus H7N9 was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates the proportion of individuals who become ill with or die from a disease in a population initially uninfected of 20% to 30% for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic , depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic.",
"The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe?",
"A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences.",
"On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse.",
"These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 david.swerdlow@pfizer.com . Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium.",
"Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB.",
"References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52 suppl 1 :S1-S3. .1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A H1N1 : a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al.",
"BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12.:687-695. .1016/S1473-3099.70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10.:1258-1263. .3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198.",
"Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198. .3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome MERS . Microbiol Spectr. 2016;4.. .1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A H7N9 as an example. Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al.",
"Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. .1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020.",
"N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness .",
"Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness . This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS.",
"When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures.",
"But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen?",
"Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever influenza-like illness .Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus.",
"Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source .",
"The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source . Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association.",
"Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo"
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What is the acronym MERS-CoV?
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Middle East respiratory syndrome coronavirus
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"Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323.:1129-1130. .1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview 25:53 COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A H1N1 virus H1N1 pdm09 pandemic or the severe acute respiratory syndrome coronavirus SARS-CoV or the Middle East respiratory syndrome coronavirus MERS-CoV epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness.",
"Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A H1N1 pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe.",
"By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe. SARS-CoV . and MERS-CoV 2012-current cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV indicating sustained and even worldwide transmission could occur , and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals.",
"SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries.",
"Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical border and school closing, infection control measures and pharmaceutical antiviral prophylaxis, vaccines interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance.",
"Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention CDC distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States.",
"Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms.",
"However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus H7N9 was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates the proportion of individuals who become ill with or die from a disease in a population initially uninfected of 20% to 30% for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic , depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic.",
"The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe?",
"A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences.",
"On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse.",
"These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 david.swerdlow@pfizer.com . Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium.",
"Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB.",
"References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52 suppl 1 :S1-S3. .1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A H1N1 : a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al.",
"BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12.:687-695. .1016/S1473-3099.70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10.:1258-1263. .3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198.",
"Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198. .3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome MERS . Microbiol Spectr. 2016;4.. .1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A H7N9 as an example. Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al.",
"Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. .1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020.",
"N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness .",
"Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness . This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS.",
"When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures.",
"But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen?",
"Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever influenza-like illness .Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus.",
"Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source .",
"The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source . Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association.",
"Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo"
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What are the critical factors that determine the effect of an epidemic?
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Transmissibility and severity
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"Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323.:1129-1130. .1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview 25:53 COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A H1N1 virus H1N1 pdm09 pandemic or the severe acute respiratory syndrome coronavirus SARS-CoV or the Middle East respiratory syndrome coronavirus MERS-CoV epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness.",
"Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A H1N1 pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe.",
"By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe. SARS-CoV . and MERS-CoV 2012-current cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV indicating sustained and even worldwide transmission could occur , and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals.",
"SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries.",
"Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical border and school closing, infection control measures and pharmaceutical antiviral prophylaxis, vaccines interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance.",
"Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention CDC distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States.",
"Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms.",
"However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus H7N9 was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates the proportion of individuals who become ill with or die from a disease in a population initially uninfected of 20% to 30% for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic , depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic.",
"The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe?",
"A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences.",
"On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse.",
"These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 david.swerdlow@pfizer.com . Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium.",
"Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB.",
"References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52 suppl 1 :S1-S3. .1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A H1N1 : a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al.",
"BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12.:687-695. .1016/S1473-3099.70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10.:1258-1263. .3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198.",
"Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198. .3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome MERS . Microbiol Spectr. 2016;4.. .1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A H7N9 as an example. Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al.",
"Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. .1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020.",
"N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness .",
"Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness . This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS.",
"When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures.",
"But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen?",
"Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever influenza-like illness .Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus.",
"Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source .",
"The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source . Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association.",
"Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo"
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When did the World Health Organization (WHO) officially declare the 2019-nCoV epidemic as a Public Health Emergency of International Concern?
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"Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323.:1129-1130. .1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview 25:53 COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A H1N1 virus H1N1 pdm09 pandemic or the severe acute respiratory syndrome coronavirus SARS-CoV or the Middle East respiratory syndrome coronavirus MERS-CoV epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness.",
"Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A H1N1 pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe.",
"By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe. SARS-CoV . and MERS-CoV 2012-current cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV indicating sustained and even worldwide transmission could occur , and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals.",
"SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries.",
"Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical border and school closing, infection control measures and pharmaceutical antiviral prophylaxis, vaccines interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance.",
"Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention CDC distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States.",
"Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms.",
"However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus H7N9 was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates the proportion of individuals who become ill with or die from a disease in a population initially uninfected of 20% to 30% for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic , depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic.",
"The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe?",
"A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences.",
"On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse.",
"These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 david.swerdlow@pfizer.com . Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium.",
"Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB.",
"References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52 suppl 1 :S1-S3. .1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A H1N1 : a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al.",
"BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12.:687-695. .1016/S1473-3099.70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10.:1258-1263. .3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198.",
"Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198. .3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome MERS . Microbiol Spectr. 2016;4.. .1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A H7N9 as an example. Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al.",
"Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. .1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020.",
"N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness .",
"Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness . This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS.",
"When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures.",
"But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen?",
"Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever influenza-like illness .Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus.",
"Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source .",
"The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source . Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association.",
"Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo"
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What influenza virus was identified in China in 2013?
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H7N9
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"Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323.:1129-1130. .1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview 25:53 COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A H1N1 virus H1N1 pdm09 pandemic or the severe acute respiratory syndrome coronavirus SARS-CoV or the Middle East respiratory syndrome coronavirus MERS-CoV epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness.",
"Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A H1N1 pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe.",
"By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe. SARS-CoV . and MERS-CoV 2012-current cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV indicating sustained and even worldwide transmission could occur , and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals.",
"SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries.",
"Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical border and school closing, infection control measures and pharmaceutical antiviral prophylaxis, vaccines interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance.",
"Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention CDC distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States.",
"Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms.",
"However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus H7N9 was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates the proportion of individuals who become ill with or die from a disease in a population initially uninfected of 20% to 30% for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic , depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic.",
"The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe?",
"A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences.",
"On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse.",
"These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 david.swerdlow@pfizer.com . Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium.",
"Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB.",
"References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52 suppl 1 :S1-S3. .1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A H1N1 : a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al.",
"BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12.:687-695. .1016/S1473-3099.70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10.:1258-1263. .3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198.",
"Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198. .3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome MERS . Microbiol Spectr. 2016;4.. .1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A H7N9 as an example. Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al.",
"Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. .1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020.",
"N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness .",
"Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness . This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS.",
"When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures.",
"But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen?",
"Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever influenza-like illness .Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus.",
"Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source .",
"The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source . Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association.",
"Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo"
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What past research has been done on severe, single-wave pandemics?
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After a new influenza virus (H7N9) was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.
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"Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323.:1129-1130. .1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview 25:53 COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A H1N1 virus H1N1 pdm09 pandemic or the severe acute respiratory syndrome coronavirus SARS-CoV or the Middle East respiratory syndrome coronavirus MERS-CoV epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness.",
"Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A H1N1 pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe.",
"By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe. SARS-CoV . and MERS-CoV 2012-current cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV indicating sustained and even worldwide transmission could occur , and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals.",
"SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries.",
"Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical border and school closing, infection control measures and pharmaceutical antiviral prophylaxis, vaccines interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance.",
"Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention CDC distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States.",
"Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms.",
"However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus H7N9 was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates the proportion of individuals who become ill with or die from a disease in a population initially uninfected of 20% to 30% for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic , depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic.",
"The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe?",
"A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences.",
"On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse.",
"These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 david.swerdlow@pfizer.com . Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium.",
"Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB.",
"References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52 suppl 1 :S1-S3. .1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A H1N1 : a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al.",
"BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12.:687-695. .1016/S1473-3099.70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10.:1258-1263. .3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198.",
"Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198. .3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome MERS . Microbiol Spectr. 2016;4.. .1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A H7N9 as an example. Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al.",
"Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. .1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020.",
"N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness .",
"Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness . This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS.",
"When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures.",
"But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen?",
"Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever influenza-like illness .Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus.",
"Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source .",
"The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source . Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association.",
"Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo"
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What is a clinical attack rate?
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the proportion of individuals who become ill with or die from a disease in a population initially uninfected
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"Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323.:1129-1130. .1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview 25:53 COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A H1N1 virus H1N1 pdm09 pandemic or the severe acute respiratory syndrome coronavirus SARS-CoV or the Middle East respiratory syndrome coronavirus MERS-CoV epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness.",
"Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A H1N1 pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe.",
"By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe. SARS-CoV . and MERS-CoV 2012-current cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV indicating sustained and even worldwide transmission could occur , and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals.",
"SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries.",
"Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical border and school closing, infection control measures and pharmaceutical antiviral prophylaxis, vaccines interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance.",
"Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention CDC distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States.",
"Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms.",
"However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus H7N9 was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates the proportion of individuals who become ill with or die from a disease in a population initially uninfected of 20% to 30% for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic , depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic.",
"The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe?",
"A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences.",
"On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse.",
"These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 david.swerdlow@pfizer.com . Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium.",
"Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB.",
"References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52 suppl 1 :S1-S3. .1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A H1N1 : a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al.",
"BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12.:687-695. .1016/S1473-3099.70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10.:1258-1263. .3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198.",
"Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198. .3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome MERS . Microbiol Spectr. 2016;4.. .1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A H7N9 as an example. Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al.",
"Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. .1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020.",
"N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness .",
"Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness . This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS.",
"When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures.",
"But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen?",
"Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever influenza-like illness .Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus.",
"Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source .",
"The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source . Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association.",
"Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo"
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What was the clinical attack rate in the 2009 H1N1 pandemic?
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"Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323.:1129-1130. .1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview 25:53 COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A H1N1 virus H1N1 pdm09 pandemic or the severe acute respiratory syndrome coronavirus SARS-CoV or the Middle East respiratory syndrome coronavirus MERS-CoV epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness.",
"Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A H1N1 pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe.",
"By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe. SARS-CoV . and MERS-CoV 2012-current cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV indicating sustained and even worldwide transmission could occur , and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals.",
"SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries.",
"Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical border and school closing, infection control measures and pharmaceutical antiviral prophylaxis, vaccines interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance.",
"Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention CDC distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States.",
"Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms.",
"However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus H7N9 was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates the proportion of individuals who become ill with or die from a disease in a population initially uninfected of 20% to 30% for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic , depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic.",
"The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe?",
"A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences.",
"On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse.",
"These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 david.swerdlow@pfizer.com . Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium.",
"Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB.",
"References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52 suppl 1 :S1-S3. .1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A H1N1 : a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al.",
"BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12.:687-695. .1016/S1473-3099.70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10.:1258-1263. .3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198.",
"Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198. .3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome MERS . Microbiol Spectr. 2016;4.. .1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A H7N9 as an example. Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al.",
"Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. .1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020.",
"N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness .",
"Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness . This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS.",
"When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures.",
"But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen?",
"Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever influenza-like illness .Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus.",
"Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source .",
"The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source . Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association.",
"Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo"
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"Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323.:1129-1130. .1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview 25:53 COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A H1N1 virus H1N1 pdm09 pandemic or the severe acute respiratory syndrome coronavirus SARS-CoV or the Middle East respiratory syndrome coronavirus MERS-CoV epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness.",
"Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A H1N1 pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe.",
"By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe. SARS-CoV . and MERS-CoV 2012-current cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV indicating sustained and even worldwide transmission could occur , and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals.",
"SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries.",
"Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical border and school closing, infection control measures and pharmaceutical antiviral prophylaxis, vaccines interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance.",
"Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention CDC distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States.",
"Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms.",
"However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus H7N9 was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates the proportion of individuals who become ill with or die from a disease in a population initially uninfected of 20% to 30% for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic , depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic.",
"The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe?",
"A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences.",
"On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse.",
"These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 david.swerdlow@pfizer.com . Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium.",
"Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB.",
"References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52 suppl 1 :S1-S3. .1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A H1N1 : a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al.",
"BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12.:687-695. .1016/S1473-3099.70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10.:1258-1263. .3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198.",
"Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198. .3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome MERS . Microbiol Spectr. 2016;4.. .1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A H7N9 as an example. Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al.",
"Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. .1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020.",
"N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness .",
"Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness . This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS.",
"When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures.",
"But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen?",
"Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever influenza-like illness .Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus.",
"Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source .",
"The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source . Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association.",
"Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo"
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How many ventilators have past studies projected will be required for a pandemic in the United States?
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"Preparation for Possible Sustained Transmission of 2019 Novel Coronavirus Lessons From Previous Epidemics February 11, 2020 David L. Swerdlow, MD1; Lyn Finelli, DrPH, MS2 Author Affiliations Article Information JAMA. 2020;323.:1129-1130. .1001/jama.2020.1960 COVID-19 Resource Center related articles icon Related Articles author interview icon Interviews Audio Interview 25:53 COVID-19 Update From China Transmissibility and severity are the 2 most critical factors that determine the effect of an epidemic. Neither the 2009 pandemic influenza A H1N1 virus H1N1 pdm09 pandemic or the severe acute respiratory syndrome coronavirus SARS-CoV or the Middle East respiratory syndrome coronavirus MERS-CoV epidemics had the combination of both high transmissibility and severity. Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness.",
"Control strategies are driven by this combination. R0, the basic reproduction number, is a commonly used measure of transmissibility and is defined as the number of additional persons one case infects over the course of their illness. An R0 of less than 1 indicates the infection will die out “eventually.” An R0 of greater than 1 indicates the infection has the potential for sustained transmission. For example, influenza A H1N1 pdm09, first identified in southern California on April 15, 2009, was highly transmissible. By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe.",
"By May 5, 2009, influenza A H1N1 pdm09 had spread to 41 US states and 21 countries.1 While influenza A H1N1 pdm09 was highly transmissible, it was not severe. Initial estimates of the R0 of influenza A H1N1 pdm09 were 1.7.2 Although an estimated 201 200 respiratory deaths due to influenza A H1N1 pdm09 occurred during the first year of the pandemic, the number of deaths per population was 30 times lower than that seen during the 1968 influenza pandemic, 1000 times less than the 1918 pandemic, and even less than typical seasonal influenza epidemics estimated by the World Health Organization WHO to be 250 000 to 500 000 per year, although estimation methods differ .3 Influenza A H1N1 pdm09 was highly transmissible but not severe. SARS-CoV . and MERS-CoV 2012-current cause severe disease, but despite the initial R0 estimations of greater than 2.0 for SARS-CoV indicating sustained and even worldwide transmission could occur , and some large outbreaks, neither were as transmissible as initial concerns suggested. SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals.",
"SARS-CoV caused 8098 reported cases and 774 deaths case-fatality rate, 9.6% in 37 countries before the epidemic was controlled. Control was thought to have been possible because a high proportion of cases were severe, making it easier to rapidly identify and isolate infected individuals. In addition, the virus was present at lower levels in upper airway secretions. There was no secondary transmission in the United States from the 8 imported cases, although in Toronto, Canada, a single importation is thought to have led to about 400 cases and 44 deaths. Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries.",
"Later estimates of R0 were less than 1, indicating that SARS-CoV may not have been capable of sustained transmission, especially in the setting of control measures.4 Similarly, MERS-CoV appears to have high severity and low transmissibility. Since 2012, MERS-CoV has caused 2494 reported cases and 858 deaths case-fatality rate, 34% in 27 countries. MERS-CoV has also caused some rapid outbreaks, mainly in hospitals in Saudi Arabia, Jordan, and South Korea, but estimates of MERS-CoV R0 are less than 1, and thus far it has been contained.5 Can a respiratory virus that is both transmissible and severe be contained? In preparation for an influenza pandemic, the US Department of Health and Human Services’ Pandemic Influenza Plan included a combination of nonpharmaceutical border and school closing, infection control measures and pharmaceutical antiviral prophylaxis, vaccines interventions meant to be used in combination to interrupt or slow influenza transmission. Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance.",
"Despite implementation of some of these interventions, influenza A H1N1 pdm09 spread to 120 countries in 3 months. With the emergence of MERS-CoV in the Middle East, a preparedness plan was developed that included a surveillance plan, laboratory testing, and contact tracing guidance. Infection control guidance was developed for use in health care settings and traveler guidance was developed for the public.6 The US Centers for Disease Control and Prevention CDC distributed MERS-CoV polymerase chain reaction test kits to state health departments. Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States.",
"Two cases were imported into the United States. Contacts were traced, including household, hospital, and airline contacts. No secondary cases were identified in the United States. MERS-CoV was thought to be severe and control measures relied on recognition of suspect cases. However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms.",
"However, during a hospital outbreak in Jeddah, Saudi Arabia, among hospitalized patients only 5 of 53 9% health care–associated cases had documented presence in the same room as a patient with MERS.5 Despite the high case-fatality rate an important measure of severity , MERS cases can be asymptomatic and mild 25% in one outbreak . Although it is not known how often asymptomatic or mildly symptomatic patients transmit MERS, initiating comprehensive measures such as isolating patients suspected of having or having been exposed to the virus and using personal protective equipment when caring for them may be extremely difficult because so many patients have mild and nonspecific symptoms. Is the world ready for a respiratory virus with high transmissibility and severity? After a new influenza virus H7N9 was identified in China in 2013, a series of modeling articles described the effect of, and level of preparedness for, a severe, single-wave pandemic in the United States.7 In scenarios that used clinical attack rates the proportion of individuals who become ill with or die from a disease in a population initially uninfected of 20% to 30% for comparison the clinical attack rate was 20% in the first year of the 2009 H1N1 pandemic , depending on severity there would be an estimated 669 000 to 4.3 million hospitalizations and an estimated 54 000 to 538 000 deaths without any interventions in the United States. The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic.",
"The models suggested that without a vaccine, school closures would be unlikely to affect the pandemic, an estimated 35 000 to 60 000 ventilators would be needed, up to an estimated 7.3 billion surgical masks or respirators would be required, and perhaps most important, if vaccine development did not start before the virus was introduced, it was unlikely that a significant number of hospitalizations and deaths could be averted due to the time it takes to develop, test, manufacture, and distribute a vaccine. It is impossible to know what will happen so early in this novel 2019 coronavirus 2019-nCoV epidemic. The scope, morbidity, and mortality will depend on the combination of severity and transmissibility. Numerous experts have “nowcasted” how many cases have occurred and forecasted how many cases will likely occur. A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe?",
"A recent study suggests rapid person to person transmission can occur.8 Disease modelers have estimated R0 to be 2.2.9 The University of Hong Kong estimates the outbreak could infect more than 150 000 persons per day in China at its peak. Is 2019-nCoV infection severe? To date approximately 14% of cases of 2019-nCoV have been described as severe by WHO, with a case-fatality rate of 2.1%.10 Estimates of severity are usually higher in the beginning of an epidemic due to the identification of the most severely affected cases and decline as the epidemic progresses. However, because many infected persons have not yet recovered and may still die, the case-fatality rate and severity could be underestimated. On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences.",
"On January 30, 2020, WHO officially declared the 2019-nCoV epidemic as a Public Health Emergency of International Concern, indicating its concern that countries aside from China could be affected by 2019-nCoV. In preparing for possible sustained transmission of 2019-nCoV beyond China, applicable lessons from previous experiences with epidemics/pandemics of respiratory viruses should be carefully considered to better control and mitigate potential consequences. Influenza preparedness plans have been developed that aim to stop, slow, or limit the spread of an influenza pandemic to the United States. These plans address limiting domestic spread and mitigating disease but also sustaining infrastructure and reducing the adverse effects of the pandemic on the economy and society. These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse.",
"These plans would be useful to enact during the 2019-nCoV epidemic should the United States experience sustained transmission. Countries have been successful in the past and there is nothing yet to predict that this time it is likely to be worse. Effective prevention and control will not be easy if there is sustained transmission and will require the full attention of public health, federal and local governments, the private sector, and every citizen. Back to topArticle Information Corresponding Author: David L. Swerdlow, MD, Clinical Epidemiology Lead, Medical Development and Scientific/Clinical Affairs, Pfizer Vaccines, 500 Arcola Rd, Collegeville, PA 19426 david.swerdlow@pfizer.com . Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium.",
"Published Online: February 11, 2020. .1001/jama.2020.1960 Conflict of Interest Disclosures: Dr Swerdlow reports owning stock and stock options in Pfizer Inc. Dr Swerdlow also reports providing a one-time consultation consisting of an overview of SARS and MERS epidemiology to GLG Consulting and receiving an honorarium. Dr Finelli reports owning stock in Merck and Co. Funding/Support: Pfizer Inc provided salary support for Dr Swerdlow. Role of the Funder/Sponsor: Pfizer Inc reviewed the manuscript and approved the decision to submit the manuscript for publication. References 1. Swerdlow DL, Finelli L, Bridges CB.",
"References 1. Swerdlow DL, Finelli L, Bridges CB. 2009 H1N1 influenza pandemic: field and epidemiologic investigations in the United States at the start of the first pandemic of the 21st century. Clin Infect Dis. 2011;52 suppl 1 :S1-S3. .1093/cid/ciq005PubMedGoogle ScholarCrossref 2. Balcan D, Hu H, Goncalves B, et al. Seasonal transmission potential and activity peaks of the new influenza A H1N1 : a Monte Carlo likelihood analysis based on human mobility. BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al.",
"BMC Medicine. 2009;7.. .1186/1741-7015-7-45 3. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. 2012;12.:687-695. .1016/S1473-3099.70121-4PubMedGoogle ScholarCrossref 4. Chowell G, Castillo-Chavez C, Fenimore PW, Kribs-Zaleta CM, Arriola L, Hyman JM. Model parameters and outbreak control for SARS. Emerg Infect Dis. 2004;10.:1258-1263. .3201/eid1007.030647PubMedGoogle ScholarCrossref 5. Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198.",
"Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East respiratory syndrome coronavirus transmission. Emerg Infect Dis. 2020;26.:191-198. .3201/eid2602.190697PubMedGoogle ScholarCrossref 6. Rasmussen SA, Watson AK, Swerdlow DL. Middle East respiratory syndrome MERS . Microbiol Spectr. 2016;4.. .1128/microbiolspec.EI10-0020-2016PubMedGoogle Scholar 7. Swerdlow DL, Pillai SK, Meltzer MI, eds. CDC modeling efforts in response to a potential public health emergency: influenza A H7N9 as an example. Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al.",
"Clin Infect Dis. 2015;60 suppl :S1-S63. Scholar 8. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus–infected pneumonia in Wuhan, China. JAMA. Published online February 7, 2020. .1001/jama.2020.1585 ArticlePubMedGoogle Scholar 9. Li Q, Guan X, Wu P, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020.",
"N Engl J Med. Published online January 29, 2020. .1056/NEJMoa2001316PubMedGoogle Scholar 10. World Health Organization. Novel coronavirus 2019-nCoV situation reports. Accessed February 4, 2020. Comment 2 Comments for this articleEXPAND ALL February 12, 2020 Understanding R and Disease Control Oz Mansoor | Public Health Physician, Wellington The message, that we need to prepare for a pandemic is vital. But the article misreports some key ideas. Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness .",
"Firstly, SARS was not controlled \"because a high proportion of cases were severe.\" While that helped , it was because cases were not infectious before some days after symptom onset usually in the second week of illness . This gave more time for case identification and isolation. And most cases did not pass on infection to anybody, but a few spread to many. When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS.",
"When all such individuals were identified and isolated, spread stopped. Unfortunately, the new virusappears to be spreading from people much earlier in the course of illness, and even with mild symptoms - which was never documented for SARS. However, it is not clear that it is any different or better at spread between people, and perhaps with the same pattern of most cases not causing further spread. Secondly, the R0, the basic reproduction number, is correctly described as the average number of infections each case causes. But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures.",
"But it lacks two key ideas: 1 the 0 after the R implies the native state, which is a fully susceptible population and without any control measures. R is the effectiive number and can include the impact of control measures. To claim that it was the lack of transmissibility, rather than the control measures that ended SARS, is not based on any evidence. And it ignores the heroic efforts of affected countries. Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen?",
"Elimination of SARS demonstrated the potential of globally coordinated collective action, as well as the damage caused by ignorance and prejudice. Most seem to have already forgotten the lessons of SARS.CONFLICT OF INTEREST: Worked for WHO/WPRO in SARS responseREAD MORE February 24, 2020 COVID 19: a global presence and not only a new pathogen? Giuliano Ramadori, Professor of Medicine | University Clinic, Göttingen, Germany In the winter season there comes the time of upper and lower respiratory tract infections characterised by cough, dyspnea and eventually fever influenza-like illness .Some of the patients, especially older people living alone affected by the disease ,may need hospitalization and eventually intensive care. In many of the cases who are hospitalized nasal and/or tracheal fluid are examined for viral or bacterial agents. Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus.",
"Only in less than 50% of the cases influenza viruses are considered to be the cause of the disease.In the rest of the cases diagnostic procedure for human coronaviruses is not performed routinely. One of the fourdifferent Human Coronaviruses HuCoV: 229E,NL 63,0C43 and HKU1 can however be found in up to 30% ofpatients negative for influenza viruses .. Chinese scientists in Wuhan, who had to deal with an increasing number of acute respiratory tract diseases resembling viral pneumonia, performed deep sequencing analysis from samples taken from the lower respiratory tract and found a \"novel\" coronavirus. The sequence of the complete genome was made public. At the same time, however, the notice from Wuhan brought to mind the SARS- and MERS-epidemics. The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source .",
"The measures taken by the Chinese- and WHO-authorities are now well known. Recently about 150 new cases have been identified in northern Italy and health authorities are still looking for case 0 the source . Is it possible that COVID-19 was already existent in Italy -- and not only in Italy but possibly everywhere in the world -- and that newly available nucleotide sequence allows now to find the cause of previously undefined influenza-like illness? REFERENCE 1. Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association.",
"Benezit F et al. :Non-influenza respiratory viruses in adult patients admitted with influenza-like illness:a 3- year prospective multicenter study.Infection, 13 february 2020, OF INTEREST: None ReportedREAD MORE See More About Global Health Public Health Pulmonary Medicine Infectious Diseases Influenza Download PDF Cite This PermissionsComment CME & MOC Coronavirus Resource Center Trending Opinion is learning has multimedia US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties March 24, 2020 Opinion is learning has multimedia 2019 Novel Coronavirus—Important Information for Clinicians March 17, 2020 Research is learning has multimedia Clinical Characteristics of Patients With Novel Coronavirus 2019-nCoV Infection Hospitalized in Beijing, China March 17, 2020 Select Your Interests JOB LISTINGS ON JAMA CAREER CENTER® ACADEMIC CARDIOLOGIST: HEART FAILURE SPECIALIST Phoenix, Arizona NONINVASIVE CARDIOLOGIST West Grove, Pennsylvania CARDIOLOGIST Phoenixville, Pennsylvania CARDIAC INTENSIVIST FACULTY West Reading, Pennsylvania CLINICAL FACULTY: CARDIOLOGY / ELECTROPHYSIOLOGIST Phoenix, Arizona See more at JAMA Career Center Others Also Liked Coronavirus Dx Emergency Use Authorizations Progressing Rapidly Despite Criticism Madeleine Johnson, 360Dx, 2020 Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Canrong Wu, Acta Pharmaceutica Sinica B, 2020 Commercial Labs Step up Coronavirus Test Efforts After FDA Guidance 360Dx, 2020 Powered by Trending US Emergency Legal Responses to Novel Coronavirus—Balancing Public Health and Civil Liberties JAMA Opinion March 24, 2020 Practical Aspects of Otolaryngologic Clinical Services During the COVID-19 Epidemic JAMA Otolaryngology–Head & Neck Surgery Opinion March 20, 2020 2019 Novel Coronavirus—Important Information for Clinicians JAMA Opinion March 17, 2020 JAMA CONTENT Home New Online Current Issue JOURNAL INFORMATION For Authors Editors & Publishers RSS Contact Us JN Learning / CME Store Apps Jobs Institutions Reprints & Permissions Journal Cover Subscribe Go JAMA Network PUBLICATIONS JAMA JAMA Network Open JAMA Cardiology JAMA Dermatology JAMA Facial Plastic Surgery JAMA Health Forum JAMA Internal Medicine JAMA Neurology JAMA Oncology JAMA Ophthalmology JAMA Otolaryngology–Head & Neck Surgery JAMA Pediatrics JAMA Psychiatry JAMA Surgery Archives of Neurology & Psychiatry 1919-1959 SITES AMA Manual of Style Art and Images in Psychiatry Breast Cancer Screening Guidelines Colorectal Screening Guidelines Declaration of Helsinki Depression Screening Guidelines Evidence-Based Medicine: An Oral History Fishbein Fellowship Genomics and Precision Health Health Disparities Hypertension Guidelines JAMA Network Audio JAMA Network Conferences Machine Learning Med Men Medical Education Opioid Management Guidelines Peer Review Congress Research Ethics Sepsis and Septic Shock Statins and Dyslipidemia Topics and Collections FEATURED ARTICLES ACS Breast Cancer Screening Guideline CDC Guideline for Prescribing Opioids CDC Guideline for Prevention of Surgical Site Infections Consensus Definitions for Sepsis and Septic Shock Global Burden of Cancer, 1990-2016 Global Burden of Disease in Children, 1990-2013 Global Burden of Hypertension, 1990-2015 Global Firearm Mortality, 1990-2016 Health Care Spending in the US and Other High-Income Countries Income and Life Expectancy in the US JNC 8 Guideline for Management of High Blood Pressure President Obama on US Health Care Reform Screening for Colorectal Cancer Screening for Depression in Adults Screening for Prostate Cancer Statins for Primary Prevention of Cardiovascular Disease The State of US Health, 1990-2016 US Burden of Cardiovascular Disease, 1990-2016 WMA Declaration of Helsinki, 7th Revision BLOGS JAMA Health Forum AMA Style Insider INFORMATION FOR Authors Institutions & Librarians Advertisers Subscription Agents Employers & Job Seekers Media JAMA NETWORK PRODUCTS AMA Manual of Style JAMAevidence JN Listen Peer Review Congress JN LEARNING Home CME Quizzes State CME Audio / Podcast Courses Clinical Challenge CME Atrial Fibrillation Course Marijuana Course Penicillin Allergy Course Cervical Cancer Screening Course CME / MOC Reporting Preferences About CME & MOC Help Subscriptions & Renewals Email Subscriptions Update Your Address Contact Us Frequently Asked Questions JAMA CAREER CENTER Physician Job Listings Get the latest from JAMA Email address Sign Up Privacy Policy | Terms of Use Jama Network Logo © 2020 American Medical Association. All Rights Reserved. Terms of Use| Privacy Policy| Accessibility Statement Silverchair Logo"
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How is exhaled breath condensate used in viral research?
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the isolation of respiratory viruses
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"BACKGROUND: Exhaled breath condensate EBC sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated.",
"The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide.",
"CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs NS , bronchoalveolar lavages BAL , nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate EBC analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease COPD , asthma, cystic fibrosis, lung cancer etc.",
"The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate EBC analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease COPD , asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli.",
"At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms defined in Table 1 , using a commercially available condenser RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA .",
"The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms defined in Table 1 , using a commercially available condenser RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA . The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven.",
"In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent Jaeger, Germany .",
"Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent Jaeger, Germany . This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs NS were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection.",
"After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit Qiagen, Westburg, The Netherlands according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer.",
"Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit Qiagen, Westburg, The Netherlands according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4 using a OneStep RT-PCR Kit Qiagen, Westburg, The Netherlands in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers Table 2 , 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer AdFW and reverse primer AdRV , 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens Table 2 . The reactions were carried out in a T3000 Thermocycler 48 Westburg, Leusden, The Netherlands with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C.",
"The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens Table 2 . The reactions were carried out in a T3000 Thermocycler 48 Westburg, Leusden, The Netherlands with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit Applied Biosystems, Foster City, CA, USA . Sequence analysis was performed with the ABI3130 Genetic Analyser Applied Biosystems, Foster City, CA, USA .",
"PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit Applied Biosystems, Foster City, CA, USA . Sequence analysis was performed with the ABI3130 Genetic Analyser Applied Biosystems, Foster City, CA, USA . Consensus sequences were obtained using the SeqMan II software DNASTAR, Madison, Wis. . For samples from HRSV was detected using a RT-PCR assay as previously described . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor Eurogentec, Seraing, Belgium 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit Ambion, Austin, TX, USA and spectrophotometrically quantified.",
"In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor Eurogentec, Seraing, Belgium 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit Ambion, Austin, TX, USA and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca HRSV-AF F 718-745 agttacacctgcattaacactaaattcc HRSV-BN N 435-458 ggctccagaatataggcatgattc HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit Ambion, Austin, TX, USA .",
"The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca HRSV-AF F 718-745 agttacacctgcattaacactaaattcc HRSV-BN N 435-458 ggctccagaatataggcatgattc HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit Ambion, Austin, TX, USA . Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System Applied Biosystems, Foster City, CA, USA . An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min.",
"The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System Applied Biosystems, Foster City, CA, USA . An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. 37.5% men, with a median age of 29 range 9 -46 years . Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old.",
"Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients 78.4% were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers 6.8% were symptomatic for 8 to 14 days and 9 participants 8.8% were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients Table 1 .",
"Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients Table 1 . Forty-seven 46.1% volunteers complained about a constant runny or stuffy nose, 43 42.2% had frequent sneezing events and 38 37.3% participants had a serious sore throat Table 1 . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses Table 2 , showed 5 RV 7.1% positive samples Table 3 . In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner.",
"Screening of the EBCs for 14 respiratory viruses Table 2 , showed 5 RV 7.1% positive samples Table 3 . In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS 46.8% , one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA subtype H1N1 and HRSV-B.",
"In 15 out of 32 NS 46.8% , one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA subtype H1N1 and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR Table 3 . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection.",
"Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in Table 3 , sample 81 was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate number of viral RNA copies exhaled per minute can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus.",
"Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc.",
"The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses.",
"With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling . In the study by Stelzer-Braid et al., 50 EBCs were analysed and St-George et al.",
"Huynh and co-workers recruited 9 volunteers for exhaled breath sampling . In the study by Stelzer-Braid et al., 50 EBCs were analysed and St-George et al. report the participation of 12 adults . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs 3 EBCs were excluded , our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus.",
"Based on the analysis of 99 EBCs 3 EBCs were excluded , our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs.",
"Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated.",
"Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection Table 3 . In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks.",
"Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection Table 3 . In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients . and NS positive patients . , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported.",
", 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC . This is in the same range of the detection rate that we report for respiratory viruses in general.",
"Recently, one study reported a detection rate of 5% for influenza in EBC . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% but the higher percentage may be due to the low number of participants subjects were included . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres . In addition, the Teflon filter has 2 μm pores which will retain all larger particles.",
"Face masks consist of electret which trap viruses based on permanently charged fibres . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are.",
"Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older . For hospitalized children a much higher rate of virus positive samples is reported .",
"The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older . For hospitalized children a much higher rate of virus positive samples is reported . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people > 70 years were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask .",
"One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections.",
"Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed.",
"In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection.",
"H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms ."
] | 1,582
| 5,193
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How many patients were i this study?
|
102
|
[
"BACKGROUND: Exhaled breath condensate EBC sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated.",
"The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide.",
"CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs NS , bronchoalveolar lavages BAL , nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate EBC analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease COPD , asthma, cystic fibrosis, lung cancer etc.",
"The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate EBC analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease COPD , asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli.",
"At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms defined in Table 1 , using a commercially available condenser RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA .",
"The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms defined in Table 1 , using a commercially available condenser RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA . The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven.",
"In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent Jaeger, Germany .",
"Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent Jaeger, Germany . This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs NS were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection.",
"After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit Qiagen, Westburg, The Netherlands according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer.",
"Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit Qiagen, Westburg, The Netherlands according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4 using a OneStep RT-PCR Kit Qiagen, Westburg, The Netherlands in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers Table 2 , 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer AdFW and reverse primer AdRV , 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens Table 2 . The reactions were carried out in a T3000 Thermocycler 48 Westburg, Leusden, The Netherlands with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C.",
"The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens Table 2 . The reactions were carried out in a T3000 Thermocycler 48 Westburg, Leusden, The Netherlands with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit Applied Biosystems, Foster City, CA, USA . Sequence analysis was performed with the ABI3130 Genetic Analyser Applied Biosystems, Foster City, CA, USA .",
"PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit Applied Biosystems, Foster City, CA, USA . Sequence analysis was performed with the ABI3130 Genetic Analyser Applied Biosystems, Foster City, CA, USA . Consensus sequences were obtained using the SeqMan II software DNASTAR, Madison, Wis. . For samples from HRSV was detected using a RT-PCR assay as previously described . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor Eurogentec, Seraing, Belgium 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit Ambion, Austin, TX, USA and spectrophotometrically quantified.",
"In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor Eurogentec, Seraing, Belgium 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit Ambion, Austin, TX, USA and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca HRSV-AF F 718-745 agttacacctgcattaacactaaattcc HRSV-BN N 435-458 ggctccagaatataggcatgattc HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit Ambion, Austin, TX, USA .",
"The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca HRSV-AF F 718-745 agttacacctgcattaacactaaattcc HRSV-BN N 435-458 ggctccagaatataggcatgattc HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit Ambion, Austin, TX, USA . Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System Applied Biosystems, Foster City, CA, USA . An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min.",
"The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System Applied Biosystems, Foster City, CA, USA . An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. 37.5% men, with a median age of 29 range 9 -46 years . Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old.",
"Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients 78.4% were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers 6.8% were symptomatic for 8 to 14 days and 9 participants 8.8% were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients Table 1 .",
"Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients Table 1 . Forty-seven 46.1% volunteers complained about a constant runny or stuffy nose, 43 42.2% had frequent sneezing events and 38 37.3% participants had a serious sore throat Table 1 . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses Table 2 , showed 5 RV 7.1% positive samples Table 3 . In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner.",
"Screening of the EBCs for 14 respiratory viruses Table 2 , showed 5 RV 7.1% positive samples Table 3 . In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS 46.8% , one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA subtype H1N1 and HRSV-B.",
"In 15 out of 32 NS 46.8% , one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA subtype H1N1 and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR Table 3 . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection.",
"Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in Table 3 , sample 81 was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate number of viral RNA copies exhaled per minute can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus.",
"Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc.",
"The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses.",
"With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling . In the study by Stelzer-Braid et al., 50 EBCs were analysed and St-George et al.",
"Huynh and co-workers recruited 9 volunteers for exhaled breath sampling . In the study by Stelzer-Braid et al., 50 EBCs were analysed and St-George et al. report the participation of 12 adults . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs 3 EBCs were excluded , our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus.",
"Based on the analysis of 99 EBCs 3 EBCs were excluded , our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs.",
"Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated.",
"Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection Table 3 . In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks.",
"Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection Table 3 . In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients . and NS positive patients . , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported.",
", 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC . This is in the same range of the detection rate that we report for respiratory viruses in general.",
"Recently, one study reported a detection rate of 5% for influenza in EBC . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% but the higher percentage may be due to the low number of participants subjects were included . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres . In addition, the Teflon filter has 2 μm pores which will retain all larger particles.",
"Face masks consist of electret which trap viruses based on permanently charged fibres . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are.",
"Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older . For hospitalized children a much higher rate of virus positive samples is reported .",
"The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older . For hospitalized children a much higher rate of virus positive samples is reported . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people > 70 years were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask .",
"One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections.",
"Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed.",
"In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection.",
"H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms ."
] | 1,582
| 5,194
|
What was the conclusion of this study?
|
EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections
|
[
"BACKGROUND: Exhaled breath condensate EBC sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated.",
"The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide.",
"CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs NS , bronchoalveolar lavages BAL , nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate EBC analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease COPD , asthma, cystic fibrosis, lung cancer etc.",
"The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate EBC analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease COPD , asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli.",
"At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms defined in Table 1 , using a commercially available condenser RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA .",
"The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms defined in Table 1 , using a commercially available condenser RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA . The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven.",
"In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent Jaeger, Germany .",
"Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent Jaeger, Germany . This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs NS were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection.",
"After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit Qiagen, Westburg, The Netherlands according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer.",
"Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit Qiagen, Westburg, The Netherlands according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4 using a OneStep RT-PCR Kit Qiagen, Westburg, The Netherlands in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers Table 2 , 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer AdFW and reverse primer AdRV , 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens Table 2 . The reactions were carried out in a T3000 Thermocycler 48 Westburg, Leusden, The Netherlands with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C.",
"The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens Table 2 . The reactions were carried out in a T3000 Thermocycler 48 Westburg, Leusden, The Netherlands with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit Applied Biosystems, Foster City, CA, USA . Sequence analysis was performed with the ABI3130 Genetic Analyser Applied Biosystems, Foster City, CA, USA .",
"PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit Applied Biosystems, Foster City, CA, USA . Sequence analysis was performed with the ABI3130 Genetic Analyser Applied Biosystems, Foster City, CA, USA . Consensus sequences were obtained using the SeqMan II software DNASTAR, Madison, Wis. . For samples from HRSV was detected using a RT-PCR assay as previously described . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor Eurogentec, Seraing, Belgium 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit Ambion, Austin, TX, USA and spectrophotometrically quantified.",
"In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor Eurogentec, Seraing, Belgium 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit Ambion, Austin, TX, USA and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca HRSV-AF F 718-745 agttacacctgcattaacactaaattcc HRSV-BN N 435-458 ggctccagaatataggcatgattc HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit Ambion, Austin, TX, USA .",
"The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca HRSV-AF F 718-745 agttacacctgcattaacactaaattcc HRSV-BN N 435-458 ggctccagaatataggcatgattc HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit Ambion, Austin, TX, USA . Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System Applied Biosystems, Foster City, CA, USA . An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min.",
"The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System Applied Biosystems, Foster City, CA, USA . An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. 37.5% men, with a median age of 29 range 9 -46 years . Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old.",
"Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients 78.4% were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers 6.8% were symptomatic for 8 to 14 days and 9 participants 8.8% were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients Table 1 .",
"Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients Table 1 . Forty-seven 46.1% volunteers complained about a constant runny or stuffy nose, 43 42.2% had frequent sneezing events and 38 37.3% participants had a serious sore throat Table 1 . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses Table 2 , showed 5 RV 7.1% positive samples Table 3 . In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner.",
"Screening of the EBCs for 14 respiratory viruses Table 2 , showed 5 RV 7.1% positive samples Table 3 . In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS 46.8% , one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA subtype H1N1 and HRSV-B.",
"In 15 out of 32 NS 46.8% , one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA subtype H1N1 and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR Table 3 . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection.",
"Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in Table 3 , sample 81 was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate number of viral RNA copies exhaled per minute can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus.",
"Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc.",
"The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses.",
"With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling . In the study by Stelzer-Braid et al., 50 EBCs were analysed and St-George et al.",
"Huynh and co-workers recruited 9 volunteers for exhaled breath sampling . In the study by Stelzer-Braid et al., 50 EBCs were analysed and St-George et al. report the participation of 12 adults . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs 3 EBCs were excluded , our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus.",
"Based on the analysis of 99 EBCs 3 EBCs were excluded , our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs.",
"Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated.",
"Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection Table 3 . In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks.",
"Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection Table 3 . In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients . and NS positive patients . , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported.",
", 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC . This is in the same range of the detection rate that we report for respiratory viruses in general.",
"Recently, one study reported a detection rate of 5% for influenza in EBC . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% but the higher percentage may be due to the low number of participants subjects were included . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres . In addition, the Teflon filter has 2 μm pores which will retain all larger particles.",
"Face masks consist of electret which trap viruses based on permanently charged fibres . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are.",
"Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older . For hospitalized children a much higher rate of virus positive samples is reported .",
"The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older . For hospitalized children a much higher rate of virus positive samples is reported . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people > 70 years were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask .",
"One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections.",
"Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed.",
"In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection.",
"H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms ."
] | 1,582
| 5,195
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How long did the patient breath into the RTube?
|
10 minutes
|
[
"BACKGROUND: Exhaled breath condensate EBC sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated.",
"The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide.",
"CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs NS , bronchoalveolar lavages BAL , nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate EBC analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease COPD , asthma, cystic fibrosis, lung cancer etc.",
"The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate EBC analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease COPD , asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli.",
"At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms defined in Table 1 , using a commercially available condenser RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA .",
"The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms defined in Table 1 , using a commercially available condenser RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA . The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven.",
"In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent Jaeger, Germany .",
"Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent Jaeger, Germany . This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs NS were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection.",
"After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit Qiagen, Westburg, The Netherlands according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer.",
"Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit Qiagen, Westburg, The Netherlands according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4 using a OneStep RT-PCR Kit Qiagen, Westburg, The Netherlands in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers Table 2 , 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer AdFW and reverse primer AdRV , 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens Table 2 . The reactions were carried out in a T3000 Thermocycler 48 Westburg, Leusden, The Netherlands with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C.",
"The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens Table 2 . The reactions were carried out in a T3000 Thermocycler 48 Westburg, Leusden, The Netherlands with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit Applied Biosystems, Foster City, CA, USA . Sequence analysis was performed with the ABI3130 Genetic Analyser Applied Biosystems, Foster City, CA, USA .",
"PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit Applied Biosystems, Foster City, CA, USA . Sequence analysis was performed with the ABI3130 Genetic Analyser Applied Biosystems, Foster City, CA, USA . Consensus sequences were obtained using the SeqMan II software DNASTAR, Madison, Wis. . For samples from HRSV was detected using a RT-PCR assay as previously described . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor Eurogentec, Seraing, Belgium 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit Ambion, Austin, TX, USA and spectrophotometrically quantified.",
"In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor Eurogentec, Seraing, Belgium 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit Ambion, Austin, TX, USA and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca HRSV-AF F 718-745 agttacacctgcattaacactaaattcc HRSV-BN N 435-458 ggctccagaatataggcatgattc HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit Ambion, Austin, TX, USA .",
"The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca HRSV-AF F 718-745 agttacacctgcattaacactaaattcc HRSV-BN N 435-458 ggctccagaatataggcatgattc HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit Ambion, Austin, TX, USA . Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System Applied Biosystems, Foster City, CA, USA . An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min.",
"The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System Applied Biosystems, Foster City, CA, USA . An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. 37.5% men, with a median age of 29 range 9 -46 years . Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old.",
"Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients 78.4% were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers 6.8% were symptomatic for 8 to 14 days and 9 participants 8.8% were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients Table 1 .",
"Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients Table 1 . Forty-seven 46.1% volunteers complained about a constant runny or stuffy nose, 43 42.2% had frequent sneezing events and 38 37.3% participants had a serious sore throat Table 1 . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses Table 2 , showed 5 RV 7.1% positive samples Table 3 . In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner.",
"Screening of the EBCs for 14 respiratory viruses Table 2 , showed 5 RV 7.1% positive samples Table 3 . In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS 46.8% , one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA subtype H1N1 and HRSV-B.",
"In 15 out of 32 NS 46.8% , one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA subtype H1N1 and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR Table 3 . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection.",
"Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in Table 3 , sample 81 was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate number of viral RNA copies exhaled per minute can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus.",
"Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc.",
"The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses.",
"With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling . In the study by Stelzer-Braid et al., 50 EBCs were analysed and St-George et al.",
"Huynh and co-workers recruited 9 volunteers for exhaled breath sampling . In the study by Stelzer-Braid et al., 50 EBCs were analysed and St-George et al. report the participation of 12 adults . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs 3 EBCs were excluded , our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus.",
"Based on the analysis of 99 EBCs 3 EBCs were excluded , our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs.",
"Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated.",
"Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection Table 3 . In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks.",
"Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection Table 3 . In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients . and NS positive patients . , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported.",
", 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC . This is in the same range of the detection rate that we report for respiratory viruses in general.",
"Recently, one study reported a detection rate of 5% for influenza in EBC . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% but the higher percentage may be due to the low number of participants subjects were included . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres . In addition, the Teflon filter has 2 μm pores which will retain all larger particles.",
"Face masks consist of electret which trap viruses based on permanently charged fibres . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are.",
"Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older . For hospitalized children a much higher rate of virus positive samples is reported .",
"The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older . For hospitalized children a much higher rate of virus positive samples is reported . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people > 70 years were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask .",
"One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections.",
"Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed.",
"In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection.",
"H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms ."
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What followed the reverse transcription step in the analysis?
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denaturation step
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"BACKGROUND: Exhaled breath condensate EBC sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated.",
"The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide.",
"CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs NS , bronchoalveolar lavages BAL , nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate EBC analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease COPD , asthma, cystic fibrosis, lung cancer etc.",
"The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate EBC analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease COPD , asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli.",
"At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms defined in Table 1 , using a commercially available condenser RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA .",
"The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms defined in Table 1 , using a commercially available condenser RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA . The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven.",
"In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent Jaeger, Germany .",
"Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent Jaeger, Germany . This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs NS were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection.",
"After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit Qiagen, Westburg, The Netherlands according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer.",
"Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit Qiagen, Westburg, The Netherlands according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4 using a OneStep RT-PCR Kit Qiagen, Westburg, The Netherlands in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers Table 2 , 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer AdFW and reverse primer AdRV , 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens Table 2 . The reactions were carried out in a T3000 Thermocycler 48 Westburg, Leusden, The Netherlands with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C.",
"The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens Table 2 . The reactions were carried out in a T3000 Thermocycler 48 Westburg, Leusden, The Netherlands with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit Applied Biosystems, Foster City, CA, USA . Sequence analysis was performed with the ABI3130 Genetic Analyser Applied Biosystems, Foster City, CA, USA .",
"PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit Applied Biosystems, Foster City, CA, USA . Sequence analysis was performed with the ABI3130 Genetic Analyser Applied Biosystems, Foster City, CA, USA . Consensus sequences were obtained using the SeqMan II software DNASTAR, Madison, Wis. . For samples from HRSV was detected using a RT-PCR assay as previously described . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor Eurogentec, Seraing, Belgium 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit Ambion, Austin, TX, USA and spectrophotometrically quantified.",
"In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor Eurogentec, Seraing, Belgium 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit Ambion, Austin, TX, USA and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca HRSV-AF F 718-745 agttacacctgcattaacactaaattcc HRSV-BN N 435-458 ggctccagaatataggcatgattc HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit Ambion, Austin, TX, USA .",
"The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca HRSV-AF F 718-745 agttacacctgcattaacactaaattcc HRSV-BN N 435-458 ggctccagaatataggcatgattc HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit Ambion, Austin, TX, USA . Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System Applied Biosystems, Foster City, CA, USA . An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min.",
"The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System Applied Biosystems, Foster City, CA, USA . An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. 37.5% men, with a median age of 29 range 9 -46 years . Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old.",
"Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients 78.4% were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers 6.8% were symptomatic for 8 to 14 days and 9 participants 8.8% were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients Table 1 .",
"Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients Table 1 . Forty-seven 46.1% volunteers complained about a constant runny or stuffy nose, 43 42.2% had frequent sneezing events and 38 37.3% participants had a serious sore throat Table 1 . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses Table 2 , showed 5 RV 7.1% positive samples Table 3 . In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner.",
"Screening of the EBCs for 14 respiratory viruses Table 2 , showed 5 RV 7.1% positive samples Table 3 . In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS 46.8% , one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA subtype H1N1 and HRSV-B.",
"In 15 out of 32 NS 46.8% , one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA subtype H1N1 and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR Table 3 . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection.",
"Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in Table 3 , sample 81 was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate number of viral RNA copies exhaled per minute can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus.",
"Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc.",
"The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses.",
"With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling . In the study by Stelzer-Braid et al., 50 EBCs were analysed and St-George et al.",
"Huynh and co-workers recruited 9 volunteers for exhaled breath sampling . In the study by Stelzer-Braid et al., 50 EBCs were analysed and St-George et al. report the participation of 12 adults . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs 3 EBCs were excluded , our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus.",
"Based on the analysis of 99 EBCs 3 EBCs were excluded , our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs.",
"Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated.",
"Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection Table 3 . In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks.",
"Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection Table 3 . In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients . and NS positive patients . , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported.",
", 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC . This is in the same range of the detection rate that we report for respiratory viruses in general.",
"Recently, one study reported a detection rate of 5% for influenza in EBC . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% but the higher percentage may be due to the low number of participants subjects were included . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres . In addition, the Teflon filter has 2 μm pores which will retain all larger particles.",
"Face masks consist of electret which trap viruses based on permanently charged fibres . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are.",
"Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older . For hospitalized children a much higher rate of virus positive samples is reported .",
"The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older . For hospitalized children a much higher rate of virus positive samples is reported . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people > 70 years were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask .",
"One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections.",
"Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed.",
"In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection.",
"H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms ."
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amplification step
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"BACKGROUND: Exhaled breath condensate EBC sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated.",
"The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide.",
"CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs NS , bronchoalveolar lavages BAL , nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate EBC analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease COPD , asthma, cystic fibrosis, lung cancer etc.",
"The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate EBC analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease COPD , asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli.",
"At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms defined in Table 1 , using a commercially available condenser RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA .",
"The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms defined in Table 1 , using a commercially available condenser RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA . The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven.",
"In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent Jaeger, Germany .",
"Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent Jaeger, Germany . This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs NS were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection.",
"After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit Qiagen, Westburg, The Netherlands according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer.",
"Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit Qiagen, Westburg, The Netherlands according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4 using a OneStep RT-PCR Kit Qiagen, Westburg, The Netherlands in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers Table 2 , 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer AdFW and reverse primer AdRV , 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens Table 2 . The reactions were carried out in a T3000 Thermocycler 48 Westburg, Leusden, The Netherlands with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C.",
"The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens Table 2 . The reactions were carried out in a T3000 Thermocycler 48 Westburg, Leusden, The Netherlands with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit Applied Biosystems, Foster City, CA, USA . Sequence analysis was performed with the ABI3130 Genetic Analyser Applied Biosystems, Foster City, CA, USA .",
"PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit Applied Biosystems, Foster City, CA, USA . Sequence analysis was performed with the ABI3130 Genetic Analyser Applied Biosystems, Foster City, CA, USA . Consensus sequences were obtained using the SeqMan II software DNASTAR, Madison, Wis. . For samples from HRSV was detected using a RT-PCR assay as previously described . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor Eurogentec, Seraing, Belgium 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit Ambion, Austin, TX, USA and spectrophotometrically quantified.",
"In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor Eurogentec, Seraing, Belgium 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit Ambion, Austin, TX, USA and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca HRSV-AF F 718-745 agttacacctgcattaacactaaattcc HRSV-BN N 435-458 ggctccagaatataggcatgattc HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit Ambion, Austin, TX, USA .",
"The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca HRSV-AF F 718-745 agttacacctgcattaacactaaattcc HRSV-BN N 435-458 ggctccagaatataggcatgattc HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit Ambion, Austin, TX, USA . Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System Applied Biosystems, Foster City, CA, USA . An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min.",
"The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System Applied Biosystems, Foster City, CA, USA . An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. 37.5% men, with a median age of 29 range 9 -46 years . Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old.",
"Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients 78.4% were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers 6.8% were symptomatic for 8 to 14 days and 9 participants 8.8% were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients Table 1 .",
"Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients Table 1 . Forty-seven 46.1% volunteers complained about a constant runny or stuffy nose, 43 42.2% had frequent sneezing events and 38 37.3% participants had a serious sore throat Table 1 . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses Table 2 , showed 5 RV 7.1% positive samples Table 3 . In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner.",
"Screening of the EBCs for 14 respiratory viruses Table 2 , showed 5 RV 7.1% positive samples Table 3 . In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS 46.8% , one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA subtype H1N1 and HRSV-B.",
"In 15 out of 32 NS 46.8% , one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA subtype H1N1 and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR Table 3 . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection.",
"Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in Table 3 , sample 81 was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate number of viral RNA copies exhaled per minute can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus.",
"Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc.",
"The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses.",
"With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling . In the study by Stelzer-Braid et al., 50 EBCs were analysed and St-George et al.",
"Huynh and co-workers recruited 9 volunteers for exhaled breath sampling . In the study by Stelzer-Braid et al., 50 EBCs were analysed and St-George et al. report the participation of 12 adults . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs 3 EBCs were excluded , our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus.",
"Based on the analysis of 99 EBCs 3 EBCs were excluded , our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs.",
"Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated.",
"Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection Table 3 . In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks.",
"Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection Table 3 . In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients . and NS positive patients . , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported.",
", 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC . This is in the same range of the detection rate that we report for respiratory viruses in general.",
"Recently, one study reported a detection rate of 5% for influenza in EBC . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% but the higher percentage may be due to the low number of participants subjects were included . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres . In addition, the Teflon filter has 2 μm pores which will retain all larger particles.",
"Face masks consist of electret which trap viruses based on permanently charged fibres . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are.",
"Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older . For hospitalized children a much higher rate of virus positive samples is reported .",
"The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older . For hospitalized children a much higher rate of virus positive samples is reported . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people > 70 years were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask .",
"One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections.",
"Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed.",
"In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection.",
"H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms ."
] | 1,582
| 5,198
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What percentage of the patients were between 20 and 30 years old in this study?
|
52%
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[
"BACKGROUND: Exhaled breath condensate EBC sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated.",
"The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide.",
"CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs NS , bronchoalveolar lavages BAL , nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate EBC analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease COPD , asthma, cystic fibrosis, lung cancer etc.",
"The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate EBC analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease COPD , asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli.",
"At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms defined in Table 1 , using a commercially available condenser RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA .",
"The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms defined in Table 1 , using a commercially available condenser RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA . The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven.",
"In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent Jaeger, Germany .",
"Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent Jaeger, Germany . This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs NS were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection.",
"After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit Qiagen, Westburg, The Netherlands according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer.",
"Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit Qiagen, Westburg, The Netherlands according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4 using a OneStep RT-PCR Kit Qiagen, Westburg, The Netherlands in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers Table 2 , 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer AdFW and reverse primer AdRV , 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens Table 2 . The reactions were carried out in a T3000 Thermocycler 48 Westburg, Leusden, The Netherlands with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C.",
"The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens Table 2 . The reactions were carried out in a T3000 Thermocycler 48 Westburg, Leusden, The Netherlands with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit Applied Biosystems, Foster City, CA, USA . Sequence analysis was performed with the ABI3130 Genetic Analyser Applied Biosystems, Foster City, CA, USA .",
"PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit Applied Biosystems, Foster City, CA, USA . Sequence analysis was performed with the ABI3130 Genetic Analyser Applied Biosystems, Foster City, CA, USA . Consensus sequences were obtained using the SeqMan II software DNASTAR, Madison, Wis. . For samples from HRSV was detected using a RT-PCR assay as previously described . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor Eurogentec, Seraing, Belgium 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit Ambion, Austin, TX, USA and spectrophotometrically quantified.",
"In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor Eurogentec, Seraing, Belgium 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit Ambion, Austin, TX, USA and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca HRSV-AF F 718-745 agttacacctgcattaacactaaattcc HRSV-BN N 435-458 ggctccagaatataggcatgattc HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit Ambion, Austin, TX, USA .",
"The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca HRSV-AF F 718-745 agttacacctgcattaacactaaattcc HRSV-BN N 435-458 ggctccagaatataggcatgattc HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit Ambion, Austin, TX, USA . Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System Applied Biosystems, Foster City, CA, USA . An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min.",
"The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System Applied Biosystems, Foster City, CA, USA . An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. 37.5% men, with a median age of 29 range 9 -46 years . Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old.",
"Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients 78.4% were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers 6.8% were symptomatic for 8 to 14 days and 9 participants 8.8% were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients Table 1 .",
"Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients Table 1 . Forty-seven 46.1% volunteers complained about a constant runny or stuffy nose, 43 42.2% had frequent sneezing events and 38 37.3% participants had a serious sore throat Table 1 . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses Table 2 , showed 5 RV 7.1% positive samples Table 3 . In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner.",
"Screening of the EBCs for 14 respiratory viruses Table 2 , showed 5 RV 7.1% positive samples Table 3 . In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS 46.8% , one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA subtype H1N1 and HRSV-B.",
"In 15 out of 32 NS 46.8% , one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA subtype H1N1 and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR Table 3 . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection.",
"Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in Table 3 , sample 81 was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate number of viral RNA copies exhaled per minute can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus.",
"Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc.",
"The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses.",
"With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling . In the study by Stelzer-Braid et al., 50 EBCs were analysed and St-George et al.",
"Huynh and co-workers recruited 9 volunteers for exhaled breath sampling . In the study by Stelzer-Braid et al., 50 EBCs were analysed and St-George et al. report the participation of 12 adults . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs 3 EBCs were excluded , our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus.",
"Based on the analysis of 99 EBCs 3 EBCs were excluded , our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs.",
"Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated.",
"Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection Table 3 . In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks.",
"Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection Table 3 . In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients . and NS positive patients . , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported.",
", 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC . This is in the same range of the detection rate that we report for respiratory viruses in general.",
"Recently, one study reported a detection rate of 5% for influenza in EBC . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% but the higher percentage may be due to the low number of participants subjects were included . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres . In addition, the Teflon filter has 2 μm pores which will retain all larger particles.",
"Face masks consist of electret which trap viruses based on permanently charged fibres . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are.",
"Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older . For hospitalized children a much higher rate of virus positive samples is reported .",
"The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older . For hospitalized children a much higher rate of virus positive samples is reported . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people > 70 years were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask .",
"One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections.",
"Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed.",
"In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection.",
"H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms ."
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Why is EBC an attractive method for screening?
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easy to perform and can be conducted in a home environment
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[
"BACKGROUND: Exhaled breath condensate EBC sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated.",
"The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide.",
"CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs NS , bronchoalveolar lavages BAL , nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate EBC analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease COPD , asthma, cystic fibrosis, lung cancer etc.",
"The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate EBC analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease COPD , asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli.",
"At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms defined in Table 1 , using a commercially available condenser RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA .",
"The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms defined in Table 1 , using a commercially available condenser RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA . The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven.",
"In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent Jaeger, Germany .",
"Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent Jaeger, Germany . This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs NS were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection.",
"After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit Qiagen, Westburg, The Netherlands according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer.",
"Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit Qiagen, Westburg, The Netherlands according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4 using a OneStep RT-PCR Kit Qiagen, Westburg, The Netherlands in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers Table 2 , 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer AdFW and reverse primer AdRV , 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens Table 2 . The reactions were carried out in a T3000 Thermocycler 48 Westburg, Leusden, The Netherlands with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C.",
"The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens Table 2 . The reactions were carried out in a T3000 Thermocycler 48 Westburg, Leusden, The Netherlands with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit Applied Biosystems, Foster City, CA, USA . Sequence analysis was performed with the ABI3130 Genetic Analyser Applied Biosystems, Foster City, CA, USA .",
"PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit Applied Biosystems, Foster City, CA, USA . Sequence analysis was performed with the ABI3130 Genetic Analyser Applied Biosystems, Foster City, CA, USA . Consensus sequences were obtained using the SeqMan II software DNASTAR, Madison, Wis. . For samples from HRSV was detected using a RT-PCR assay as previously described . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor Eurogentec, Seraing, Belgium 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit Ambion, Austin, TX, USA and spectrophotometrically quantified.",
"In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor Eurogentec, Seraing, Belgium 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit Ambion, Austin, TX, USA and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca HRSV-AF F 718-745 agttacacctgcattaacactaaattcc HRSV-BN N 435-458 ggctccagaatataggcatgattc HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit Ambion, Austin, TX, USA .",
"The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca HRSV-AF F 718-745 agttacacctgcattaacactaaattcc HRSV-BN N 435-458 ggctccagaatataggcatgattc HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit Ambion, Austin, TX, USA . Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System Applied Biosystems, Foster City, CA, USA . An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min.",
"The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System Applied Biosystems, Foster City, CA, USA . An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. 37.5% men, with a median age of 29 range 9 -46 years . Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old.",
"Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients 78.4% were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers 6.8% were symptomatic for 8 to 14 days and 9 participants 8.8% were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients Table 1 .",
"Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients Table 1 . Forty-seven 46.1% volunteers complained about a constant runny or stuffy nose, 43 42.2% had frequent sneezing events and 38 37.3% participants had a serious sore throat Table 1 . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses Table 2 , showed 5 RV 7.1% positive samples Table 3 . In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner.",
"Screening of the EBCs for 14 respiratory viruses Table 2 , showed 5 RV 7.1% positive samples Table 3 . In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS 46.8% , one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA subtype H1N1 and HRSV-B.",
"In 15 out of 32 NS 46.8% , one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA subtype H1N1 and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR Table 3 . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection.",
"Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in Table 3 , sample 81 was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate number of viral RNA copies exhaled per minute can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus.",
"Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc.",
"The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses.",
"With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling . In the study by Stelzer-Braid et al., 50 EBCs were analysed and St-George et al.",
"Huynh and co-workers recruited 9 volunteers for exhaled breath sampling . In the study by Stelzer-Braid et al., 50 EBCs were analysed and St-George et al. report the participation of 12 adults . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs 3 EBCs were excluded , our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus.",
"Based on the analysis of 99 EBCs 3 EBCs were excluded , our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs.",
"Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated.",
"Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection Table 3 . In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks.",
"Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection Table 3 . In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients . and NS positive patients . , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported.",
", 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC . This is in the same range of the detection rate that we report for respiratory viruses in general.",
"Recently, one study reported a detection rate of 5% for influenza in EBC . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% but the higher percentage may be due to the low number of participants subjects were included . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres . In addition, the Teflon filter has 2 μm pores which will retain all larger particles.",
"Face masks consist of electret which trap viruses based on permanently charged fibres . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are.",
"Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older . For hospitalized children a much higher rate of virus positive samples is reported .",
"The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older . For hospitalized children a much higher rate of virus positive samples is reported . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people > 70 years were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask .",
"One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections.",
"Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed.",
"In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection.",
"H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms ."
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What is the leading cause of death among children after the age of 1 month?
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Pneumonia remains the leading cause of death in children outside the neonatal period,
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[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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How has the number of childhood pneumonia been reduced?
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New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What percentage of childhood deaths are due to pneumonia?
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approximately 900,000 of the estimated 6.3 million child deaths in 2013
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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How has the childhood population grown in the last two decades?
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global childhood population from 605 million in 2000 to 664 million in 2015
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[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What is the reduction in the number of childhood pneumonia cases?
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Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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How much is the reduction in the childhood pneumonia deaths?
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Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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Childhood pneumonia rate for high income countries vs low and middle income countries.
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The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries.
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What percentage of childhood pneumonia deaths occur outside hospital in low and middle income countries?
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up to 81% of severe pneumonia deaths occur outside a hospital
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[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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Case Fatality Rates for Childhood Pneumonia in high income vs low and middle income countries.
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the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries
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[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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How can childhood pneumonia affect the subsequent health of a person?
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Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What is the increase in the risk of respiratory disease after having childhood pneumonia.
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The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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Which is the best method to identify pneumonia in a person?
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Chest radiologic changes have been considered the gold standard for defining a pneumonia event
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What is responsible for the reduction of radiologic pneumonia?
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Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia.
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What is the percentage reduction in pneumonia cases due to vaccination?
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Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What is the revised WHO definition of Bacterial Pneumonia?
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A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
] | 1,571
| 522
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What is the reduction in bacterial pneumonia under the revised WHO definition of bacterial pneumonia?
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Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa
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[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
] | 1,571
| 524
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What caused the increase in the incidence of empyema in children in the recent past?
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An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A
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[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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How have the incidence Empyema been reduced?
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These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What pneumonia related or chest conditions indicate the need for child radiography?
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chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications.
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What chest diseases and pneumonia were identified as leading causes prior to the availability of vaccines?
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prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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Why has pertussis immunity in infants has decreased in infants?
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Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What is the effect of childhood tuberculosis in childhood pneumonia?
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A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What are the risk factors in childhood pneumonia?
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incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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How does air pollution affect the incidence of childhood pneumonia?
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Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
] | 1,571
| 538
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What is the strongest risk factor for childhood pneumonia?
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The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children
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[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
] | 1,571
| 539
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What is the global coverage of influenza and pneumonia vaccines?
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By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations
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[
"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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Is influenza vaccination during pregnancy safe? How long does it protect the child?
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Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh and 50.4% in South Africa ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What is emphyema?
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a rare complication of pneumonia
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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How is the term end point consolidation described with regard to pneumonia diagnosis?
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as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.
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"Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades.",
"Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions.",
"Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study .",
"Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries .",
"Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease.",
"In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia.",
"A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major restrictive lung disease, obstructive lung disease, bronchiectasis and minor chronic bronchitis, asthma, abnormal pulmonary function groups . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung.",
"In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's WHO standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia . The term \"end-point consolidation\" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. \"Other infiltrate\" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. \"Primary end-point pneumonia\" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate including \"other\" infiltrate . Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia .",
"Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia , 25% in South Africa and 26% in the Philippines . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability.",
"The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia . A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination .",
"A revised case definition of \"presumed bacterial pneumonia\" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination . Using the revised definition, the 10-valent pneumococcal conjugate vaccine pneumococcal conjugate vaccination-10 , had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus HIV in South Africa . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A .",
"Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period 1998 to 2004 to the post-pneumococcal conjugate vaccination-7 period 2005 to 2010 . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13.",
"In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia .",
"Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases tuberculosis, inhaled foreign body or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases.",
"In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate .",
"Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa , while viruses were detected in 81% of radiologic pneumonia cases in Sweden . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls.",
"A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels.",
"In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection .",
"In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 . However recent evidence from South Africa where the acellular vaccine is used shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas.",
"A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries.",
"Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age 4.5 , stunting 2.6 and wasting 2.8 . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available .",
"Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality .",
"Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations .",
"However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa .",
"Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection vaccine efficacy 63% in Bangladesh and 50.4% in South Africa . However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden.",
"Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission .",
"Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe.",
"If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing .",
"However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed and others are ongoing . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia.",
"Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial . This has been reflected in British and Spanish national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research .",
"Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia.",
"Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines against Haemophilus influenzae and pneumococcus , have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia."
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What factors make H5N1 a worldwide threat to public health?
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rapid spread and high pathogenicity
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"The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide EAP against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection.",
"We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor MR . These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS .",
"Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome SARS . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths a mortality rate >50% , from 2003 to 2013 influenza/human animal interface/H5N1 cumulative table archives/en/index.html . Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation .",
"Several antiinfluenza medications have been widely used, including zanamivir Relenza and oseltamivir Tamiflu . Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests .",
"This plant first invaded southern Yunnan Province China in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP .",
"In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide EAP . The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza.",
"In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010.",
"Virus. The H5N1 influenza virus A/bar-headed goose/ Qinghai/1/2010 used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney MDCK cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water.",
"8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories Beijing, China , and the original breeding pairs were purchased from Charles River Beijing, China . Mice were raised in independent ventilated cages IVC and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney MDCK cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China.",
"The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator.",
"The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol v/v and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method .",
"The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide yield = 1.2% . EAP content was determined by the phenol-H 2 SO 4 method . Vitro. 2.5 mL A549 and Raw 264.7 cells 4 × 10 5 /mL per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP 50, 100, 200 g/mL was added to each well. Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR .",
"Controls were treated with phosphate-buffered saline PBS . Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction qPCR . Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA.",
"Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile Virbac, France intramuscularly at 15 mg/kg body weight and then infected intranasally with 120 plaqueforming units PFU of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay.",
"Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM Hyclone Laboratories, Logan, UT, USA containing 10% FBS Hyclone Laboratories , 100 U/mL penicillin, and 100 g/mL streptomycin Invitrogen, San Diego, CA, USA . Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet.",
"Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK Sigma-Aldrich, St. Louis, MO, USA . Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol Invitrogen according to the manufacturer's instructions. DNaseItreated RNA 0.2 g was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 .",
"The expression of the hemagglutinin HA gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit Applied Biosystems, Foster City, CA, USA . The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 Applied Biosystems using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard .",
"The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls naive mice by 2 −ΔΔCT using -actin as an internal standard . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm.",
"IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits Boster, Wuhan, China according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 SPSS Taiwan Corp., Taiwan , and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells.",
"Many botanical polysaccharides exhibit an immunomodulatory effect . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP 50, 100, 200 g/mL for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU.",
"Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells Figures 1 a -1 c and Raw264.7 cells Figures 1 d -1 f compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 a , all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant.",
"Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS by log rank analysis . EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice Figure 2 b . To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d .",
"To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP 25 mg/kg group were significantly lower than the titers in the mice that received PBS at day 3 postinfection Figures 2 c and 2 d . These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c .",
"To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group 25 mg/kg were significantly higher than those in the PBS control naive mice Figures 3 a -3 c . Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group = 0.0599 Figures 3 g -3 i . These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f .",
"IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP 25 mg/kg treatment were significantly lower than those in the PBS group Figure 3 e , while IL-6 and IFN-expression were only slightly lower not significant Figures 3 d and 3 f . These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d .",
"To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls Figures 4 c and 4 d . The lungs of EAP 25 mg/kg treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels Figures 4 a and 4 b . Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs .",
"Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors PRRs . To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 TLR2 , TLR4, Dectin-1, and mannose receptor MR were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a .",
"Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant Figure 5 a . In vitro assay showed similar trends. As shown in Figure 5 b , Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model.",
"These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro.",
"The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators IL-6, TNFand IFN- before infection. Furthermore, the effect of EAP on PRR expression including TLR2, TLR4, Dectin-1, and MR was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza .",
"Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus including H1N1 and H3N2 subtypes . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro.",
"The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment 25 mg/kg body weight was significantly higher than in mice receiving PBS 50% to 0% . In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury.",
"In previous reports, high levels of proinflammatory cytokines and chemokines including TNF-, IL-6 and IFN- were detected during H5N1 infection . This \"cytokine storm\" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect . However, it is unclear whether the lack of proinflammatory cytokines such as TNFand IL-6 facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections .",
"Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive .",
"Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage data not show . The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival .",
"The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival . TNF-has been reported to exert a defensive effect against influenza infection in vitro . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS.",
"In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response.",
"In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro.",
"In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection.",
"Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products."
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