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Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Pathogen resistance'.	A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient. Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains. 1. Case Presentation We report the case of a 12-year-old boy (35.5 kg), who received double lung transplantation in 25 March 2017, (at the age of 12) because of cystic fibrosis. Before transplantation, he was colonized with Aspergillus fumigatus, Achromobacter xylosoxidans (first colonization identified in 2013), and Pseudomonas aeruginosa. After transplantation, he experienced acute kidney injury requiring temporary haemodialysis, pulmonary embolism, and persisting airway colonization with A. fumigatus. He was discharged on 29 April 2017, with satisfactory respiratory status; he initially required no home oxygen therapy. His immunosuppressive regimen included prednisolone (25 mg/day), tacrolimus (1.5 mg bis in die (b.i.d.) with a target trough concentration of 6–8 ng/mL), mycophenolate mofetil (1 g b.i.d.) and his antimicrobial prophylaxis was cotrimoxazole (400/80 mg, three times a week), posaconazole (150 mg, oral suspension, ter in die (t.i.d.)), valacyclovir (500 mg b.i.d.), and intravenous immunoglobulins (15 g each three weeks). Between May and June 2017, he experienced progressive shortness of breath, cough and increased sputum production; he subsequently required the addition of 1–1.5 L/min oxygen therapy. Because of a stenosis of the surgical anastomosis of the main left bronchus, a dilatation during rigid bronchoscopy was performed on 19 May and a Montgomery stent was inserted on 2 June. Later on, a stenosis of the surgical anastomosis on the right bronchus intermedius required a dilatation during rigid bronchoscopy followed by the insertion of an Oki stent on 29 June. Several bronchoalveolar lavages (BAL), performed between May and June 2017, revealed inflammation (between 800 and >1000 leukocytes/mm3 with 88–90% of polymorphonuclear cells) and repeatedly grew pandrug-resistant A. xylosoxidans (Table 1 and Supplementary Figure S1) [1]. Lung biopsy performed on 19 June was consistent with lung infection: diffuse acute bronchiolitis with extension to peri-bronchiolar alveoli. Microbiological samples obtained from lung biopsies were negative (bacterial, mycological, and mycobacteriological cultures). The first antibiotic treatment consisted of intravenous tigecycline 50 mg b.i.d. from 29 June to 31 July. As BAL remained positive for A. xylosoxidans and the patient still required oxygen therapy; antibiotic treatment was subsequently switched to imipenem (1200 mg, t.i.d., ~100 mg/Kg/d) on 31 July 2017. Subsequent respiratory samples still grew A. xylosoxidans and the respiratory status did not improve (requiring oxygen at home, 1 L/min). Furthermore, despite antibiotic treatment and correct drainage of the right superior lobe, lung consolidation and micronodules remained. Considering the failure of antibiotic treatment, phage therapy was proposed to the patient and his family, after multidisciplinary discussion. A first cocktail (APC 1.1) containing three lytic phages (JWDelta, JWT and 2-1) active against A. xylosoxidans isolate is1S (designated such as later), selected from the DSMZ collection (Braunschweig, Germany) [2,3], was prepared at the Laboratory for Bacteriology Research (Ghent University, Belgium) and the Laboratory for Molecular and Cellular Technology (Queen Astrid Military Hospital, Brussels, Belgium) using the above-mentioned bacterial isolate, as previously described [4]. The production was completed on 23 August with a final phage titer of 4 × 1010 plaque forming units (pfu)/mL defined by double-agar overlay method [4], an endotoxin level of 4000 EU/mL (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, GenScript, Piscataway Township, NJ, USA) and a pH of 7.3 (in sterile PBS). Regulatory permission for phage importation was obtained on 1 September 2017, from the Agence Nationale de Sécurité du Médicament (ANSM). A first round of phage administration was performed, consisting of 3 nebulizations/day of 5 mL of the filter-sterilized (Sartorius 0.2 µm) phage solution, tenfold diluted in sterile saline on 8 and 9 September, using a vibrating mesh nebulizer (eFLOW rapid, PARI, PARI GmbH, Germany) with a mouth piece. Immediate tolerance was perfect, but no clinical improvement was noted and culture of subsequent respiratory samples remained positive for A. xylosoxidans (104 CFU/mL in the BAL of 15 September). A second cocktail (APC 2.1) was produced, in which phage JWalpha was added to the three lytic phages of cocktail APC 1.1, to improve the therapeutic efficacy. The production was completed on 22 December with a final phage titer of 5 × 109 pfu/mL, an endotoxin level of 1760 EU/mL and a pH of 6.95 (in sterile PBS). Regulatory permission was obtained for phage importation on 16 January 2018 (ANSM). On 23 January, during therapeutic bronchoscopy under general anesthesia, 30 mL of APC2.1, tenfold diluted (in sterile saline) and filter-sterilized, was instilled in each pulmonary lobe through the fibroscope. Immediate tolerance was perfect and the patient was discharged on 24 January with continued phage nebulization at home: three times a day 5 mL of preparation A using a vibrating mesh nebulizer (eFLOW rapid, PARI) with a mouth piece for 14 days until 6 February. Clinical tolerance was perfect again, but the initial clinical status remained unchanged. BAL performed on 8 February 2018, two days after stopping the phage treatment, still grew 105 cfu/mL of A. xylosoxidans. Subsequently, the patient’s respiratory condition slowly improved and oxygen therapy was stopped on 15 February 2018. No clinical worsening was observed after imipenem interruption on 16 February 2018. Sputum culture remained positive for A. xylosoxidans but with a low bacterial density (103 CFU/mL in March and May 2018, and June 2019) and no A. xylosoxidans could be isolated from BALs#10 and #11, sampled on August 2019, and April 2020, respectively (Table 1). The last pulmonary function tests, performed on 17 October 2019, showed best results since lung transplantation, with an FEV1 of 79% and a forced vital capacity of 85%. 2. Microbiological Analysis of Eight A. xylosoxidans Isolates As we observed discrepant outcome in our patient (clinical improvement on the one hand but persisting low-grade airway colonization on the other hand), we decided to study the eight available A. xylosoxidans isolates (is1 to is9) from the patient’s colonization obtained by random picking from the culture plates from 4 distinct BALs (Table 2). These eight isolates, taken before, during and after the phage therapy period (Table 2), could not be differentiated from each other by means of MALDI-TOF [5] or McRAPD typing [6] and all belonged to the epidemic clone, already described previously among Belgian CF patients [5]. Whole genome sequencing (WGS) and de novo genome assembly for these eight isolates was carried out (Supplementary Methods and Table S1). The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Total genome length for all eight isolates was 6.44–6.50 Mbp, with a GC content of 67.5%. All eight isolates showed most similarity with reference genome GCF_008432465 (AX1), which corresponds to the type strain of A. xylosoxidans subsp. xylosoxidans (ATCC 27061T, CCUG 12689T, DSMZ 10346T, LMG 1863T, and NCTC 10807T), i.e., between 88.38 and 89.60% on the basis of mapped sequence reads, and between 98.71% and 98.76% on the basis of average nucleotide identity (ANI). Indeed, the overall similarity between the genome sequences of the eight isolates was high, i.e., 99.60%, as assessed by digital DNA hybridization, and more than 99.97% according to ANI-analysis. This was confirmed by variant calling where we observed that the isolates differed from each other only for a total of 367 SNPs, whereas a total of 59,452 SNPs were observed between the isolates and the best matching reference genome (AX1) (Figure 1). The susceptibility phenotype of each of the eight isolates was assessed by spot-test against the phage cocktail APC 2.1 and expressed as “S” when susceptible or “R” when resistant (e.g., “is2S” means that isolate number 2 was susceptible to the phage cocktail) (Table 2 and Supplementary Methods). Among these eight isolates, analysis based on variant calling furthermore revealed four closely related but clearly delineated strains, i.e., clones that differ genetically from other clones, with different phage susceptibility phenotype (Table 2 and Figure 1): Strain 1 (Str1), contained isolates 1 and 3 (is1S and is3R), found in BAL#3 (is1S-Str1/BAL#3) and BAL#5 (is3R-Str1/BAL#5), respectively. Strain 2 (Str2) contained isolate 2 (is2S) from BAL#5 (is2S-Str2/BAL#5). Strain 3 (Str3) contained isolates 5, 6, and 9 (is5S, is6S and is9S), which were found in BAL#6 (is5S-Str3/BAL#6, is6S-Str3/BAL#6) and BAL#9 (is9S-Str3/BAL#9). Strain 4 contained isolates 7 and 8 (is7R and is8R), found in BAL#9 (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) (Table 2). Isolate is1S-Str1/BAL#3 was obtained on 25 July 2017, i.e., 45 days before the first phage therapy session (8 September 2017) and was 100% identical to is3R-Str1/BAL#5 isolated seven days (15 September 2017) after the first phage therapy session (Figure 1). Since both isolates is1S-Str1/BAL#3 and is3R-Str1/BAL#5 are 100% identical, this means that the phage resistance mechanism of isolate is3R-Str1/BAL#5 is not genetically supported (see discussion). Isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6, both phage susceptible, were obtained one month later (12 October 2017) and belonged to still another strain (Strain 3). Finally, on 8 February 2018, i.e., 16 days after the second phage therapy session (23 January 2018), an additional three isolates were obtained from BAL#9. Of these, isolate is9S-Str3/BAL#9 belonged to the same Strain 3 as isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6 and was phage susceptible as well, but isolates 7R and 8R belonged to still another strain (Strain 4) and were both resistant to the phage cocktail. Interestingly, the phage resistant Strain 4 isolates (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) contained mutations compared to the other six isolates that could explain the phage resistance observed for these two isolates (Figure 1): when Strain 1 was used as a reference, a C to A transition at position 1803 of the colicin I receptor gene caused a Tyr601->Stop transition. This receptor has been recognized as a phage receptor [7]. We further studied the course of infection by extracting DNA from the three available A. xylosoxidans-positive BAL samples (BAL#5, #6 and #9), and by quantifying DNA of the different strains by means of strain-specific qPCRs (Table 3 and Supplementary Methods) that had been developed on the basis of the obtained WGS data. qPCRs for Strain 2 and Strain 4 were only positive for the BALs from which these isolates had been cultured, respectively, BAL#5 and BAL#9. qPCR for Strain 1 was only positive for BAL#9, but not for BAL#5 from which isolate 3R had been cultured. qPCR for Strain 3 was positive for all three BALs, although no isolate belonging to Strain 3 was cultured from BAL#5. 3. Discussion and Conclusions We report the first case of decolonization of pandrug-resistant Achromobacter xylosoxidans after a long-term follow-up in a lung-transplanted patient receiving phage therapy. The first actual demonstration and usage of bacteriophages came almost simultaneously from two microbiologists in 1915 (Frederick Twort) and 1917 (Félix d’Hérelle) [8]. After extensive clinical use during the first half of the 20th century, their use has been progressively reduced, mainly due to the discovery, rapid spread, and ease of use of antibiotics for the treatment of infectious diseases [9]. However, bacteriophages gained a new surge of attention, due to the emergence and spread of pandrug-resistant bacteria [8]. Despite decades of clinical experience with bacteriophages in Eastern Europe, very few data are available regarding their clinical efficacy; besides, case-series and few randomized controlled trials (RCT) [10,11,12]. For instance, a RCT assessed the clinical outcome of patients receiving local application of a phage cocktail for the treatment of burn wound infection. This RCT is frequently cited to have shown no clinical benefits, as compared to standard of care. However, the phage titer that was applied (100 pfu/mL) [10], was so extremely low that it precludes to draw any conclusion from this study regarding the usefulness of phage therapy. The limited number of randomized clinical trials has been recently reviewed [13]. Phage therapy has also been used locally, e.g., for the treatment of bone and joint infections [14,15] or intravenously for the treatment of bloodstream infections [16,17]. Considering the high frequency of colonization and infection caused by antibiotic-resistant bacteria among cystic fibrosis and lung-transplanted patients, such as in our patient, the use of bacteriophages appears to be an appealing option in this population [18,19,20]. In the specific case of A. xylosoxidans, thus far a single case-report described the favorable outcome after the use of bacteriophages in a cystic fibrosis patient [21]. Our case-report highlights the many difficulties that need to be taken into account before considering the use of bacteriophages in a clinical setting. These include amongst others an important delay from clinical decision to regulatory agency authorization. Most problematic is the difficult interpretation of the clinical outcome after bacteriophage therapy, considering the long-term persisting airway colonization and the possible evolution of different A. xylosoxidans strains. The second therapeutic attempt relied on a massive lung instillation (injection of the bacteriophage solution in the patient’s lung during fibroscopy) followed by a two-week course of phage nebulization. We used a vibrating mesh nebulizer for phage delivery as this type of nebulizer has been shown to result in a lower phage titer reduction, as compared to jet nebulizers [22]. BAL#9, performed two weeks after the second round of phage therapy (8 February 2018) still grew 105 CFU/mL of A. xylosoxidans, represented by Strains 3 and 4 (according to culture) and by at least Strains 1, 3 and 4 (according to qPCR) (Table 1, Table 2 and Table 3). Sputum cultures remained positive for 18 months, but with only 103 CFU A. xylosoxidans/mL, suggesting a possible upper airway colonization, especially since no A. xylosoxidans could be isolated from BALs sampled in August 2019, and April 2020. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. To more precisely understand the discrepant outcome of our patient, another option would have been to search for active phages from the BAL samples throughout the study, but this was not performed. Despite the observed differences of a total of 367 SNPs between the four strains of eight A. xylosoxidans isolates from the patient, their overall similarity suggests that they originate from a single strain that colonized the patient (who was colonized with A. xylosoxidans years before the start of the phage therapy). Therefore, it is possible that some variants were present already before lung transplantation and before the start of phage therapy. Although it is not possible to exclude the simultaneous presence of these different strains on the basis of our culture results, because only one or two colonies were picked from each BAL culture, analysis by means of four strain-specific qPCR assays of the three BAL samples that were available for study, indicated that Strains 2 and 4 were probably prevalent at a particular moment of the course of infection, whereas Strains 1 and 3 could be identified during the full course of the infection. Phage resistant Strain 4 may have been selected during phage therapy. Phage training has been proposed to overcome phage resistance and increase infectivity against a given bacterial population [23]. However, training phages may take weeks, and therefore might be restricted to the treatment of chronic infections. Regarding more acute infections, the establishment of phage collections (such as the DSMZ, which provided bacteriophages for our patient) is of critical importance to provide phages active against a broad spectrum of bacterial isolates. An interesting point is the observation of two isolates from the same strain (is1S-Str1/BAL#3 and is3R-Str1/BAL#5) that differ regarding their phage susceptibility phenotype despite the absence of genetic support (identical genome). As observed with bacteria where emerging data point out that a resistance phenotype against a given antimicrobial may not be supported by a genotypic substratum [24,25], phage resistance without any change in genotype has also been documented [26]. These genotype/phenotype discrepancies are hypothesized to be possible through epigenetic regulation mechanisms, of which post-transcriptional regulation, which may influence phage receptor expression on the cell surface or phage receptor masking (through capsule synthesis). Further studies are required to better understand the dynamics of in vivo selection of phage-resistant bacteria. Because the patient’s medical condition improved anyhow, a few weeks after the second round of phage instillation, it is possible that phage therapy selected the phage-resistant A. xylosoxidans Strain 4, but that the mutation that caused phage resistance at the same time decreased the virulence of the strain as already described with other bacteria and phages [27,28,29]. Indeed, we observed several mutations which could explain the phage resistance of Strain 4, of which one was especially interesting, since it caused the gain of a stop codon in the colicin I receptor, which has been recognized as a phage receptor in other species [7]. Because in E. coli, the colicin I receptor plays a crucial role in iron transport across the outer membrane [7], loss of this function due to the observed mutation may have caused reduced fitness of the A. xylosoxidans mutant Strain 4. However, from a clinical point-of-view, one cannot exclude a spontaneous improvement, independently of phage therapy and despite months of fruitless antibiotic treatment, highlighting the dire need of randomized clinical trials in order to more convincingly assess the clinical efficacy of bacteriophages. In conclusion, we describe the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. Despite initial persisting airway colonization, the final clinical and microbiological outcome was favorable. Whole genome sequencing of eight A. xylosoxidans isolates and strain-specific qPCR experiments performed on BALs sampled before and after phage therapy allowed to more precisely study the dynamics of lung colonization by different isolates, susceptible or resistant to the applied phage cocktails. Acknowledgments The authors would like to thank L. Lécuyer and V. Boussaud (Unité de Transplantation, HEGP), B. Sabatier, V. Savoldelli, T. Caruba and P. Prognon (Pharmacie HEGP), P. Odou, C. Nassar, C. Berneron and M. Roche (Pharmacie Lille), and E. Deberranger (Hématologie Pédiatrique Lille), for their help in the management of the patient. Microbiological analysis was performed with a funding by the Association Robert Debré pour la Recherche Médicale. The authors would also like to thank Caroline Semaille and Nathalie Morgensztejn from the Agence Nationale de Sécurité du Médicament (ANSM) for their regulatory support. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Materials The following are available online at https://www.mdpi.com/1999-4915/13/1/60/s1, Figure S1: Antibiotic susceptibility testing (AST) of a pandrug-resistant Achromobacter xylosoxidans isolated during a bronchoalveolar lavage of a 12-year-old boy after double lung transplantation, Table S1: Variant calling for the eight Achromobacter xylosoxidans whole genome sequences, see excel file attached, named: “Supplementary Table S1. Variant calling.xls”. Click here for additional data file. Author Contributions D.L. (David Lebeaux) and M.V. conceptualized the study, validated the data, carried out formal analysis, prepared the initial draft, took care of project administration and supervised the study. Data were visualized by H.D. and Y.G. Software analysis was carried out by H.D., S.V., Y.G. D.L. (David Lebeaux), M.M., R.G., I.P., F.C., N.K., J.W., C.R., E.C., D.L. (Damien Lanoy), L.V.S., H.D., J.-P.P., N.D., S.V., Y.G., F.V.N., and M.V. contributed to basic methodology. Data curation was done by D.L. (David Lebeaux), M.M., C.T., S.V., Y.G., and M.V. Funding was acquired by D.L. (David Lebeaux), J.-L.M., F.V.N., and M.V. All authors took part in the investigation and in final reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement The study did not require ethical approval. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Phylogenetic tree built with Whole genome sequences of eight Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. The distance between samples reflects the number of different Single Nucleotide Polymorphisms (SNPs). Each A. xylosoxidans is named as follows: isolate number (is1 to is8) S (or R to depict susceptibility or resistance to phage cocktail)-Strain number/Number of BAL where it has been identified. viruses-13-00060-t001_Table 1Table 1 Results of bronchoalveolar lavages (BALs) and sputum analysis from May 2017, to April 2020, in 12-year-old lung transplanted patient who received two rounds of phage therapy for pandrug-resistant Achromobacter xylosoxidans colonization and infection. Date 19 May 2017 23 May 2017 6 June 2017 12 June 2017 25 July 2017 5 September 2017 8 September 2017 15 September 2017 12 October 2017 27 October 2017 23 January 2018 8 February 2018 5 March 2018 18 May 2018 19 February 2019 19 June 2019 27 August 2019 30 April 2020 Phage therapy ROUND 1 ROUND 2 Tigecycline from 29 June to 31 July Imipenem From 31 July 2017, to 16 February 2018 Number of days, relative to the first round of phage therapy −112 −108 −94 −88 −45 −3 +7 +37 +49 +137 +153 +178 +252 +529 +649 +718 +963 BAL BAL Number * #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 Leukocytes (/mm3) >1000 800 >1000 500 >1000 60 >1000 810 >1000 NA NA NA PMN cells (%) 90 88 90 91 43 30 80 69 82 NA NA NA Quantification of A. x culture (CFU/mL) 104 103 104 103 104 103 104 106 105 104 0 0 Other bacteria (CFU/mL) None None None None None None None None None None 104 S. a 106 S. a Sputum Leucocytes (/field) >25 >25 >25 >25 NA NA NA Quantification of A. x culture (CFU/mL) 105 105 105 106 103 103 103 Other bacteria (CFU/mL) None None None None 108 M. c None 106 S. a A. x: Achromobacter xylosoxidans; M. c: Moraxella catarrhalis; NA: non available; PMN: Polymorphonuclear; RBC: red blood cells; S. a: Staphylococcus aureus. : BAL number in bold indicates BALs for which qPCR had been carried out. viruses-13-00060-t002_Table 2Table 2 Eight Achromobacter xylosoxidans isolates for which whole genome sequencing was carried out. These were isolated from bronchoalveolar lavages in a 12-year-old boy after double lung transplantation. The patient received a first round of phage administration on 8 September 2017. A second round of phage treatment was performed on 23 January 2018. Complete Name Isolate Strain Source of Sampling Date of Sampling Whole Genome Sequence is1S-Str1/BAL#3 is1S Str1 BAL#3 25 July 2017 ERS5044236-UGAX1 is2S-Str2/BAL#5 is2S Str2 BAL#5 15 September 2017 ERS5044237-UGAX2 is3R-Str1/BAL#5 is3R Str1 BAL#5 15 September 2017 ERS5044238-UGAX3 is5S-Str3/BAL#6 is5S Str3 BAL#6 12 October 2017 ERS5044239-UGAX5 is6S-Str3/BAL#6 is6S Str3 BAL#6 12 October 2017 ERS5044240-UGAX6 is7R-Str4/BAL#9 is7R Str4 BAL#9 8 February 2018 ERS5044241-UGAX7 is8R-Str4/BAL#9 is8R Str4 BAL#9 8 February 2018 ERS5044242-UGAX8 is9S-Str3/BAL#9 is9S Str3 BAL#9 8 February 2018 ERS5044243-UGAX9 * Submitted at ENA, 1 September 2020. Study ID: PRJEB39103. BAL: Bronchoalveolar lavage, S: Phage susceptible, R: phage resistant. viruses-13-00060-t003_Table 3Table 3 Results of bacterial culture (grey-shading) and qPCR of three broncho-alveolar lavages for four different Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. Bronchoalveolar Lavage BAL#5 BAL#6 BAL#9 15 September 2017 12 October 2017 8 February 2018 qPCR for Strain Isolates Str1 is1S, is3R N NS P (34.7) Str2 is2S P (36.6) N N Str3 is5S, is6S, is9S P (32.7) P (28.9) P (27.7) Str4 is7R, is8R NS NS P (35.2) Grey-shaded box = isolate cultured from this BAL; N: negative (Cq-value > 40); NS: Nonspecific amplification (melting peak different from that of specific amplification); P: positive qPCR (Cq-value).	MYCOPHENOLATE MOFETIL, POSACONAZOLE, PREDNISOLONE, SULFAMETHOXAZOLE\TRIMETHOPRIM, TACROLIMUS, VALACYCLOVIR HYDROCHLORIDE	DrugsGivenReaction	CC BY	33466377	19,957,163	2021-01-05
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Pneumonia bacterial'.	A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient. Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains. 1. Case Presentation We report the case of a 12-year-old boy (35.5 kg), who received double lung transplantation in 25 March 2017, (at the age of 12) because of cystic fibrosis. Before transplantation, he was colonized with Aspergillus fumigatus, Achromobacter xylosoxidans (first colonization identified in 2013), and Pseudomonas aeruginosa. After transplantation, he experienced acute kidney injury requiring temporary haemodialysis, pulmonary embolism, and persisting airway colonization with A. fumigatus. He was discharged on 29 April 2017, with satisfactory respiratory status; he initially required no home oxygen therapy. His immunosuppressive regimen included prednisolone (25 mg/day), tacrolimus (1.5 mg bis in die (b.i.d.) with a target trough concentration of 6–8 ng/mL), mycophenolate mofetil (1 g b.i.d.) and his antimicrobial prophylaxis was cotrimoxazole (400/80 mg, three times a week), posaconazole (150 mg, oral suspension, ter in die (t.i.d.)), valacyclovir (500 mg b.i.d.), and intravenous immunoglobulins (15 g each three weeks). Between May and June 2017, he experienced progressive shortness of breath, cough and increased sputum production; he subsequently required the addition of 1–1.5 L/min oxygen therapy. Because of a stenosis of the surgical anastomosis of the main left bronchus, a dilatation during rigid bronchoscopy was performed on 19 May and a Montgomery stent was inserted on 2 June. Later on, a stenosis of the surgical anastomosis on the right bronchus intermedius required a dilatation during rigid bronchoscopy followed by the insertion of an Oki stent on 29 June. Several bronchoalveolar lavages (BAL), performed between May and June 2017, revealed inflammation (between 800 and >1000 leukocytes/mm3 with 88–90% of polymorphonuclear cells) and repeatedly grew pandrug-resistant A. xylosoxidans (Table 1 and Supplementary Figure S1) [1]. Lung biopsy performed on 19 June was consistent with lung infection: diffuse acute bronchiolitis with extension to peri-bronchiolar alveoli. Microbiological samples obtained from lung biopsies were negative (bacterial, mycological, and mycobacteriological cultures). The first antibiotic treatment consisted of intravenous tigecycline 50 mg b.i.d. from 29 June to 31 July. As BAL remained positive for A. xylosoxidans and the patient still required oxygen therapy; antibiotic treatment was subsequently switched to imipenem (1200 mg, t.i.d., ~100 mg/Kg/d) on 31 July 2017. Subsequent respiratory samples still grew A. xylosoxidans and the respiratory status did not improve (requiring oxygen at home, 1 L/min). Furthermore, despite antibiotic treatment and correct drainage of the right superior lobe, lung consolidation and micronodules remained. Considering the failure of antibiotic treatment, phage therapy was proposed to the patient and his family, after multidisciplinary discussion. A first cocktail (APC 1.1) containing three lytic phages (JWDelta, JWT and 2-1) active against A. xylosoxidans isolate is1S (designated such as later), selected from the DSMZ collection (Braunschweig, Germany) [2,3], was prepared at the Laboratory for Bacteriology Research (Ghent University, Belgium) and the Laboratory for Molecular and Cellular Technology (Queen Astrid Military Hospital, Brussels, Belgium) using the above-mentioned bacterial isolate, as previously described [4]. The production was completed on 23 August with a final phage titer of 4 × 1010 plaque forming units (pfu)/mL defined by double-agar overlay method [4], an endotoxin level of 4000 EU/mL (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, GenScript, Piscataway Township, NJ, USA) and a pH of 7.3 (in sterile PBS). Regulatory permission for phage importation was obtained on 1 September 2017, from the Agence Nationale de Sécurité du Médicament (ANSM). A first round of phage administration was performed, consisting of 3 nebulizations/day of 5 mL of the filter-sterilized (Sartorius 0.2 µm) phage solution, tenfold diluted in sterile saline on 8 and 9 September, using a vibrating mesh nebulizer (eFLOW rapid, PARI, PARI GmbH, Germany) with a mouth piece. Immediate tolerance was perfect, but no clinical improvement was noted and culture of subsequent respiratory samples remained positive for A. xylosoxidans (104 CFU/mL in the BAL of 15 September). A second cocktail (APC 2.1) was produced, in which phage JWalpha was added to the three lytic phages of cocktail APC 1.1, to improve the therapeutic efficacy. The production was completed on 22 December with a final phage titer of 5 × 109 pfu/mL, an endotoxin level of 1760 EU/mL and a pH of 6.95 (in sterile PBS). Regulatory permission was obtained for phage importation on 16 January 2018 (ANSM). On 23 January, during therapeutic bronchoscopy under general anesthesia, 30 mL of APC2.1, tenfold diluted (in sterile saline) and filter-sterilized, was instilled in each pulmonary lobe through the fibroscope. Immediate tolerance was perfect and the patient was discharged on 24 January with continued phage nebulization at home: three times a day 5 mL of preparation A using a vibrating mesh nebulizer (eFLOW rapid, PARI) with a mouth piece for 14 days until 6 February. Clinical tolerance was perfect again, but the initial clinical status remained unchanged. BAL performed on 8 February 2018, two days after stopping the phage treatment, still grew 105 cfu/mL of A. xylosoxidans. Subsequently, the patient’s respiratory condition slowly improved and oxygen therapy was stopped on 15 February 2018. No clinical worsening was observed after imipenem interruption on 16 February 2018. Sputum culture remained positive for A. xylosoxidans but with a low bacterial density (103 CFU/mL in March and May 2018, and June 2019) and no A. xylosoxidans could be isolated from BALs#10 and #11, sampled on August 2019, and April 2020, respectively (Table 1). The last pulmonary function tests, performed on 17 October 2019, showed best results since lung transplantation, with an FEV1 of 79% and a forced vital capacity of 85%. 2. Microbiological Analysis of Eight A. xylosoxidans Isolates As we observed discrepant outcome in our patient (clinical improvement on the one hand but persisting low-grade airway colonization on the other hand), we decided to study the eight available A. xylosoxidans isolates (is1 to is9) from the patient’s colonization obtained by random picking from the culture plates from 4 distinct BALs (Table 2). These eight isolates, taken before, during and after the phage therapy period (Table 2), could not be differentiated from each other by means of MALDI-TOF [5] or McRAPD typing [6] and all belonged to the epidemic clone, already described previously among Belgian CF patients [5]. Whole genome sequencing (WGS) and de novo genome assembly for these eight isolates was carried out (Supplementary Methods and Table S1). The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Total genome length for all eight isolates was 6.44–6.50 Mbp, with a GC content of 67.5%. All eight isolates showed most similarity with reference genome GCF_008432465 (AX1), which corresponds to the type strain of A. xylosoxidans subsp. xylosoxidans (ATCC 27061T, CCUG 12689T, DSMZ 10346T, LMG 1863T, and NCTC 10807T), i.e., between 88.38 and 89.60% on the basis of mapped sequence reads, and between 98.71% and 98.76% on the basis of average nucleotide identity (ANI). Indeed, the overall similarity between the genome sequences of the eight isolates was high, i.e., 99.60%, as assessed by digital DNA hybridization, and more than 99.97% according to ANI-analysis. This was confirmed by variant calling where we observed that the isolates differed from each other only for a total of 367 SNPs, whereas a total of 59,452 SNPs were observed between the isolates and the best matching reference genome (AX1) (Figure 1). The susceptibility phenotype of each of the eight isolates was assessed by spot-test against the phage cocktail APC 2.1 and expressed as “S” when susceptible or “R” when resistant (e.g., “is2S” means that isolate number 2 was susceptible to the phage cocktail) (Table 2 and Supplementary Methods). Among these eight isolates, analysis based on variant calling furthermore revealed four closely related but clearly delineated strains, i.e., clones that differ genetically from other clones, with different phage susceptibility phenotype (Table 2 and Figure 1): Strain 1 (Str1), contained isolates 1 and 3 (is1S and is3R), found in BAL#3 (is1S-Str1/BAL#3) and BAL#5 (is3R-Str1/BAL#5), respectively. Strain 2 (Str2) contained isolate 2 (is2S) from BAL#5 (is2S-Str2/BAL#5). Strain 3 (Str3) contained isolates 5, 6, and 9 (is5S, is6S and is9S), which were found in BAL#6 (is5S-Str3/BAL#6, is6S-Str3/BAL#6) and BAL#9 (is9S-Str3/BAL#9). Strain 4 contained isolates 7 and 8 (is7R and is8R), found in BAL#9 (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) (Table 2). Isolate is1S-Str1/BAL#3 was obtained on 25 July 2017, i.e., 45 days before the first phage therapy session (8 September 2017) and was 100% identical to is3R-Str1/BAL#5 isolated seven days (15 September 2017) after the first phage therapy session (Figure 1). Since both isolates is1S-Str1/BAL#3 and is3R-Str1/BAL#5 are 100% identical, this means that the phage resistance mechanism of isolate is3R-Str1/BAL#5 is not genetically supported (see discussion). Isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6, both phage susceptible, were obtained one month later (12 October 2017) and belonged to still another strain (Strain 3). Finally, on 8 February 2018, i.e., 16 days after the second phage therapy session (23 January 2018), an additional three isolates were obtained from BAL#9. Of these, isolate is9S-Str3/BAL#9 belonged to the same Strain 3 as isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6 and was phage susceptible as well, but isolates 7R and 8R belonged to still another strain (Strain 4) and were both resistant to the phage cocktail. Interestingly, the phage resistant Strain 4 isolates (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) contained mutations compared to the other six isolates that could explain the phage resistance observed for these two isolates (Figure 1): when Strain 1 was used as a reference, a C to A transition at position 1803 of the colicin I receptor gene caused a Tyr601->Stop transition. This receptor has been recognized as a phage receptor [7]. We further studied the course of infection by extracting DNA from the three available A. xylosoxidans-positive BAL samples (BAL#5, #6 and #9), and by quantifying DNA of the different strains by means of strain-specific qPCRs (Table 3 and Supplementary Methods) that had been developed on the basis of the obtained WGS data. qPCRs for Strain 2 and Strain 4 were only positive for the BALs from which these isolates had been cultured, respectively, BAL#5 and BAL#9. qPCR for Strain 1 was only positive for BAL#9, but not for BAL#5 from which isolate 3R had been cultured. qPCR for Strain 3 was positive for all three BALs, although no isolate belonging to Strain 3 was cultured from BAL#5. 3. Discussion and Conclusions We report the first case of decolonization of pandrug-resistant Achromobacter xylosoxidans after a long-term follow-up in a lung-transplanted patient receiving phage therapy. The first actual demonstration and usage of bacteriophages came almost simultaneously from two microbiologists in 1915 (Frederick Twort) and 1917 (Félix d’Hérelle) [8]. After extensive clinical use during the first half of the 20th century, their use has been progressively reduced, mainly due to the discovery, rapid spread, and ease of use of antibiotics for the treatment of infectious diseases [9]. However, bacteriophages gained a new surge of attention, due to the emergence and spread of pandrug-resistant bacteria [8]. Despite decades of clinical experience with bacteriophages in Eastern Europe, very few data are available regarding their clinical efficacy; besides, case-series and few randomized controlled trials (RCT) [10,11,12]. For instance, a RCT assessed the clinical outcome of patients receiving local application of a phage cocktail for the treatment of burn wound infection. This RCT is frequently cited to have shown no clinical benefits, as compared to standard of care. However, the phage titer that was applied (100 pfu/mL) [10], was so extremely low that it precludes to draw any conclusion from this study regarding the usefulness of phage therapy. The limited number of randomized clinical trials has been recently reviewed [13]. Phage therapy has also been used locally, e.g., for the treatment of bone and joint infections [14,15] or intravenously for the treatment of bloodstream infections [16,17]. Considering the high frequency of colonization and infection caused by antibiotic-resistant bacteria among cystic fibrosis and lung-transplanted patients, such as in our patient, the use of bacteriophages appears to be an appealing option in this population [18,19,20]. In the specific case of A. xylosoxidans, thus far a single case-report described the favorable outcome after the use of bacteriophages in a cystic fibrosis patient [21]. Our case-report highlights the many difficulties that need to be taken into account before considering the use of bacteriophages in a clinical setting. These include amongst others an important delay from clinical decision to regulatory agency authorization. Most problematic is the difficult interpretation of the clinical outcome after bacteriophage therapy, considering the long-term persisting airway colonization and the possible evolution of different A. xylosoxidans strains. The second therapeutic attempt relied on a massive lung instillation (injection of the bacteriophage solution in the patient’s lung during fibroscopy) followed by a two-week course of phage nebulization. We used a vibrating mesh nebulizer for phage delivery as this type of nebulizer has been shown to result in a lower phage titer reduction, as compared to jet nebulizers [22]. BAL#9, performed two weeks after the second round of phage therapy (8 February 2018) still grew 105 CFU/mL of A. xylosoxidans, represented by Strains 3 and 4 (according to culture) and by at least Strains 1, 3 and 4 (according to qPCR) (Table 1, Table 2 and Table 3). Sputum cultures remained positive for 18 months, but with only 103 CFU A. xylosoxidans/mL, suggesting a possible upper airway colonization, especially since no A. xylosoxidans could be isolated from BALs sampled in August 2019, and April 2020. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. To more precisely understand the discrepant outcome of our patient, another option would have been to search for active phages from the BAL samples throughout the study, but this was not performed. Despite the observed differences of a total of 367 SNPs between the four strains of eight A. xylosoxidans isolates from the patient, their overall similarity suggests that they originate from a single strain that colonized the patient (who was colonized with A. xylosoxidans years before the start of the phage therapy). Therefore, it is possible that some variants were present already before lung transplantation and before the start of phage therapy. Although it is not possible to exclude the simultaneous presence of these different strains on the basis of our culture results, because only one or two colonies were picked from each BAL culture, analysis by means of four strain-specific qPCR assays of the three BAL samples that were available for study, indicated that Strains 2 and 4 were probably prevalent at a particular moment of the course of infection, whereas Strains 1 and 3 could be identified during the full course of the infection. Phage resistant Strain 4 may have been selected during phage therapy. Phage training has been proposed to overcome phage resistance and increase infectivity against a given bacterial population [23]. However, training phages may take weeks, and therefore might be restricted to the treatment of chronic infections. Regarding more acute infections, the establishment of phage collections (such as the DSMZ, which provided bacteriophages for our patient) is of critical importance to provide phages active against a broad spectrum of bacterial isolates. An interesting point is the observation of two isolates from the same strain (is1S-Str1/BAL#3 and is3R-Str1/BAL#5) that differ regarding their phage susceptibility phenotype despite the absence of genetic support (identical genome). As observed with bacteria where emerging data point out that a resistance phenotype against a given antimicrobial may not be supported by a genotypic substratum [24,25], phage resistance without any change in genotype has also been documented [26]. These genotype/phenotype discrepancies are hypothesized to be possible through epigenetic regulation mechanisms, of which post-transcriptional regulation, which may influence phage receptor expression on the cell surface or phage receptor masking (through capsule synthesis). Further studies are required to better understand the dynamics of in vivo selection of phage-resistant bacteria. Because the patient’s medical condition improved anyhow, a few weeks after the second round of phage instillation, it is possible that phage therapy selected the phage-resistant A. xylosoxidans Strain 4, but that the mutation that caused phage resistance at the same time decreased the virulence of the strain as already described with other bacteria and phages [27,28,29]. Indeed, we observed several mutations which could explain the phage resistance of Strain 4, of which one was especially interesting, since it caused the gain of a stop codon in the colicin I receptor, which has been recognized as a phage receptor in other species [7]. Because in E. coli, the colicin I receptor plays a crucial role in iron transport across the outer membrane [7], loss of this function due to the observed mutation may have caused reduced fitness of the A. xylosoxidans mutant Strain 4. However, from a clinical point-of-view, one cannot exclude a spontaneous improvement, independently of phage therapy and despite months of fruitless antibiotic treatment, highlighting the dire need of randomized clinical trials in order to more convincingly assess the clinical efficacy of bacteriophages. In conclusion, we describe the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. Despite initial persisting airway colonization, the final clinical and microbiological outcome was favorable. Whole genome sequencing of eight A. xylosoxidans isolates and strain-specific qPCR experiments performed on BALs sampled before and after phage therapy allowed to more precisely study the dynamics of lung colonization by different isolates, susceptible or resistant to the applied phage cocktails. Acknowledgments The authors would like to thank L. Lécuyer and V. Boussaud (Unité de Transplantation, HEGP), B. Sabatier, V. Savoldelli, T. Caruba and P. Prognon (Pharmacie HEGP), P. Odou, C. Nassar, C. Berneron and M. Roche (Pharmacie Lille), and E. Deberranger (Hématologie Pédiatrique Lille), for their help in the management of the patient. Microbiological analysis was performed with a funding by the Association Robert Debré pour la Recherche Médicale. The authors would also like to thank Caroline Semaille and Nathalie Morgensztejn from the Agence Nationale de Sécurité du Médicament (ANSM) for their regulatory support. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Materials The following are available online at https://www.mdpi.com/1999-4915/13/1/60/s1, Figure S1: Antibiotic susceptibility testing (AST) of a pandrug-resistant Achromobacter xylosoxidans isolated during a bronchoalveolar lavage of a 12-year-old boy after double lung transplantation, Table S1: Variant calling for the eight Achromobacter xylosoxidans whole genome sequences, see excel file attached, named: “Supplementary Table S1. Variant calling.xls”. Click here for additional data file. Author Contributions D.L. (David Lebeaux) and M.V. conceptualized the study, validated the data, carried out formal analysis, prepared the initial draft, took care of project administration and supervised the study. Data were visualized by H.D. and Y.G. Software analysis was carried out by H.D., S.V., Y.G. D.L. (David Lebeaux), M.M., R.G., I.P., F.C., N.K., J.W., C.R., E.C., D.L. (Damien Lanoy), L.V.S., H.D., J.-P.P., N.D., S.V., Y.G., F.V.N., and M.V. contributed to basic methodology. Data curation was done by D.L. (David Lebeaux), M.M., C.T., S.V., Y.G., and M.V. Funding was acquired by D.L. (David Lebeaux), J.-L.M., F.V.N., and M.V. All authors took part in the investigation and in final reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement The study did not require ethical approval. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Phylogenetic tree built with Whole genome sequences of eight Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. The distance between samples reflects the number of different Single Nucleotide Polymorphisms (SNPs). Each A. xylosoxidans is named as follows: isolate number (is1 to is8) S (or R to depict susceptibility or resistance to phage cocktail)-Strain number/Number of BAL where it has been identified. viruses-13-00060-t001_Table 1Table 1 Results of bronchoalveolar lavages (BALs) and sputum analysis from May 2017, to April 2020, in 12-year-old lung transplanted patient who received two rounds of phage therapy for pandrug-resistant Achromobacter xylosoxidans colonization and infection. Date 19 May 2017 23 May 2017 6 June 2017 12 June 2017 25 July 2017 5 September 2017 8 September 2017 15 September 2017 12 October 2017 27 October 2017 23 January 2018 8 February 2018 5 March 2018 18 May 2018 19 February 2019 19 June 2019 27 August 2019 30 April 2020 Phage therapy ROUND 1 ROUND 2 Tigecycline from 29 June to 31 July Imipenem From 31 July 2017, to 16 February 2018 Number of days, relative to the first round of phage therapy −112 −108 −94 −88 −45 −3 +7 +37 +49 +137 +153 +178 +252 +529 +649 +718 +963 BAL BAL Number * #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 Leukocytes (/mm3) >1000 800 >1000 500 >1000 60 >1000 810 >1000 NA NA NA PMN cells (%) 90 88 90 91 43 30 80 69 82 NA NA NA Quantification of A. x culture (CFU/mL) 104 103 104 103 104 103 104 106 105 104 0 0 Other bacteria (CFU/mL) None None None None None None None None None None 104 S. a 106 S. a Sputum Leucocytes (/field) >25 >25 >25 >25 NA NA NA Quantification of A. x culture (CFU/mL) 105 105 105 106 103 103 103 Other bacteria (CFU/mL) None None None None 108 M. c None 106 S. a A. x: Achromobacter xylosoxidans; M. c: Moraxella catarrhalis; NA: non available; PMN: Polymorphonuclear; RBC: red blood cells; S. a: Staphylococcus aureus. : BAL number in bold indicates BALs for which qPCR had been carried out. viruses-13-00060-t002_Table 2Table 2 Eight Achromobacter xylosoxidans isolates for which whole genome sequencing was carried out. These were isolated from bronchoalveolar lavages in a 12-year-old boy after double lung transplantation. The patient received a first round of phage administration on 8 September 2017. A second round of phage treatment was performed on 23 January 2018. Complete Name Isolate Strain Source of Sampling Date of Sampling Whole Genome Sequence is1S-Str1/BAL#3 is1S Str1 BAL#3 25 July 2017 ERS5044236-UGAX1 is2S-Str2/BAL#5 is2S Str2 BAL#5 15 September 2017 ERS5044237-UGAX2 is3R-Str1/BAL#5 is3R Str1 BAL#5 15 September 2017 ERS5044238-UGAX3 is5S-Str3/BAL#6 is5S Str3 BAL#6 12 October 2017 ERS5044239-UGAX5 is6S-Str3/BAL#6 is6S Str3 BAL#6 12 October 2017 ERS5044240-UGAX6 is7R-Str4/BAL#9 is7R Str4 BAL#9 8 February 2018 ERS5044241-UGAX7 is8R-Str4/BAL#9 is8R Str4 BAL#9 8 February 2018 ERS5044242-UGAX8 is9S-Str3/BAL#9 is9S Str3 BAL#9 8 February 2018 ERS5044243-UGAX9 * Submitted at ENA, 1 September 2020. Study ID: PRJEB39103. BAL: Bronchoalveolar lavage, S: Phage susceptible, R: phage resistant. viruses-13-00060-t003_Table 3Table 3 Results of bacterial culture (grey-shading) and qPCR of three broncho-alveolar lavages for four different Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. Bronchoalveolar Lavage BAL#5 BAL#6 BAL#9 15 September 2017 12 October 2017 8 February 2018 qPCR for Strain Isolates Str1 is1S, is3R N NS P (34.7) Str2 is2S P (36.6) N N Str3 is5S, is6S, is9S P (32.7) P (28.9) P (27.7) Str4 is7R, is8R NS NS P (35.2) Grey-shaded box = isolate cultured from this BAL; N: negative (Cq-value > 40); NS: Nonspecific amplification (melting peak different from that of specific amplification); P: positive qPCR (Cq-value).	MYCOPHENOLATE MOFETIL, POSACONAZOLE, PREDNISOLONE, SULFAMETHOXAZOLE\TRIMETHOPRIM, TACROLIMUS, VALACYCLOVIR HYDROCHLORIDE	DrugsGivenReaction	CC BY	33466377	19,957,163	2021-01-05
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Staphylococcal infection'.	A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient. Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains. 1. Case Presentation We report the case of a 12-year-old boy (35.5 kg), who received double lung transplantation in 25 March 2017, (at the age of 12) because of cystic fibrosis. Before transplantation, he was colonized with Aspergillus fumigatus, Achromobacter xylosoxidans (first colonization identified in 2013), and Pseudomonas aeruginosa. After transplantation, he experienced acute kidney injury requiring temporary haemodialysis, pulmonary embolism, and persisting airway colonization with A. fumigatus. He was discharged on 29 April 2017, with satisfactory respiratory status; he initially required no home oxygen therapy. His immunosuppressive regimen included prednisolone (25 mg/day), tacrolimus (1.5 mg bis in die (b.i.d.) with a target trough concentration of 6–8 ng/mL), mycophenolate mofetil (1 g b.i.d.) and his antimicrobial prophylaxis was cotrimoxazole (400/80 mg, three times a week), posaconazole (150 mg, oral suspension, ter in die (t.i.d.)), valacyclovir (500 mg b.i.d.), and intravenous immunoglobulins (15 g each three weeks). Between May and June 2017, he experienced progressive shortness of breath, cough and increased sputum production; he subsequently required the addition of 1–1.5 L/min oxygen therapy. Because of a stenosis of the surgical anastomosis of the main left bronchus, a dilatation during rigid bronchoscopy was performed on 19 May and a Montgomery stent was inserted on 2 June. Later on, a stenosis of the surgical anastomosis on the right bronchus intermedius required a dilatation during rigid bronchoscopy followed by the insertion of an Oki stent on 29 June. Several bronchoalveolar lavages (BAL), performed between May and June 2017, revealed inflammation (between 800 and >1000 leukocytes/mm3 with 88–90% of polymorphonuclear cells) and repeatedly grew pandrug-resistant A. xylosoxidans (Table 1 and Supplementary Figure S1) [1]. Lung biopsy performed on 19 June was consistent with lung infection: diffuse acute bronchiolitis with extension to peri-bronchiolar alveoli. Microbiological samples obtained from lung biopsies were negative (bacterial, mycological, and mycobacteriological cultures). The first antibiotic treatment consisted of intravenous tigecycline 50 mg b.i.d. from 29 June to 31 July. As BAL remained positive for A. xylosoxidans and the patient still required oxygen therapy; antibiotic treatment was subsequently switched to imipenem (1200 mg, t.i.d., ~100 mg/Kg/d) on 31 July 2017. Subsequent respiratory samples still grew A. xylosoxidans and the respiratory status did not improve (requiring oxygen at home, 1 L/min). Furthermore, despite antibiotic treatment and correct drainage of the right superior lobe, lung consolidation and micronodules remained. Considering the failure of antibiotic treatment, phage therapy was proposed to the patient and his family, after multidisciplinary discussion. A first cocktail (APC 1.1) containing three lytic phages (JWDelta, JWT and 2-1) active against A. xylosoxidans isolate is1S (designated such as later), selected from the DSMZ collection (Braunschweig, Germany) [2,3], was prepared at the Laboratory for Bacteriology Research (Ghent University, Belgium) and the Laboratory for Molecular and Cellular Technology (Queen Astrid Military Hospital, Brussels, Belgium) using the above-mentioned bacterial isolate, as previously described [4]. The production was completed on 23 August with a final phage titer of 4 × 1010 plaque forming units (pfu)/mL defined by double-agar overlay method [4], an endotoxin level of 4000 EU/mL (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, GenScript, Piscataway Township, NJ, USA) and a pH of 7.3 (in sterile PBS). Regulatory permission for phage importation was obtained on 1 September 2017, from the Agence Nationale de Sécurité du Médicament (ANSM). A first round of phage administration was performed, consisting of 3 nebulizations/day of 5 mL of the filter-sterilized (Sartorius 0.2 µm) phage solution, tenfold diluted in sterile saline on 8 and 9 September, using a vibrating mesh nebulizer (eFLOW rapid, PARI, PARI GmbH, Germany) with a mouth piece. Immediate tolerance was perfect, but no clinical improvement was noted and culture of subsequent respiratory samples remained positive for A. xylosoxidans (104 CFU/mL in the BAL of 15 September). A second cocktail (APC 2.1) was produced, in which phage JWalpha was added to the three lytic phages of cocktail APC 1.1, to improve the therapeutic efficacy. The production was completed on 22 December with a final phage titer of 5 × 109 pfu/mL, an endotoxin level of 1760 EU/mL and a pH of 6.95 (in sterile PBS). Regulatory permission was obtained for phage importation on 16 January 2018 (ANSM). On 23 January, during therapeutic bronchoscopy under general anesthesia, 30 mL of APC2.1, tenfold diluted (in sterile saline) and filter-sterilized, was instilled in each pulmonary lobe through the fibroscope. Immediate tolerance was perfect and the patient was discharged on 24 January with continued phage nebulization at home: three times a day 5 mL of preparation A using a vibrating mesh nebulizer (eFLOW rapid, PARI) with a mouth piece for 14 days until 6 February. Clinical tolerance was perfect again, but the initial clinical status remained unchanged. BAL performed on 8 February 2018, two days after stopping the phage treatment, still grew 105 cfu/mL of A. xylosoxidans. Subsequently, the patient’s respiratory condition slowly improved and oxygen therapy was stopped on 15 February 2018. No clinical worsening was observed after imipenem interruption on 16 February 2018. Sputum culture remained positive for A. xylosoxidans but with a low bacterial density (103 CFU/mL in March and May 2018, and June 2019) and no A. xylosoxidans could be isolated from BALs#10 and #11, sampled on August 2019, and April 2020, respectively (Table 1). The last pulmonary function tests, performed on 17 October 2019, showed best results since lung transplantation, with an FEV1 of 79% and a forced vital capacity of 85%. 2. Microbiological Analysis of Eight A. xylosoxidans Isolates As we observed discrepant outcome in our patient (clinical improvement on the one hand but persisting low-grade airway colonization on the other hand), we decided to study the eight available A. xylosoxidans isolates (is1 to is9) from the patient’s colonization obtained by random picking from the culture plates from 4 distinct BALs (Table 2). These eight isolates, taken before, during and after the phage therapy period (Table 2), could not be differentiated from each other by means of MALDI-TOF [5] or McRAPD typing [6] and all belonged to the epidemic clone, already described previously among Belgian CF patients [5]. Whole genome sequencing (WGS) and de novo genome assembly for these eight isolates was carried out (Supplementary Methods and Table S1). The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Total genome length for all eight isolates was 6.44–6.50 Mbp, with a GC content of 67.5%. All eight isolates showed most similarity with reference genome GCF_008432465 (AX1), which corresponds to the type strain of A. xylosoxidans subsp. xylosoxidans (ATCC 27061T, CCUG 12689T, DSMZ 10346T, LMG 1863T, and NCTC 10807T), i.e., between 88.38 and 89.60% on the basis of mapped sequence reads, and between 98.71% and 98.76% on the basis of average nucleotide identity (ANI). Indeed, the overall similarity between the genome sequences of the eight isolates was high, i.e., 99.60%, as assessed by digital DNA hybridization, and more than 99.97% according to ANI-analysis. This was confirmed by variant calling where we observed that the isolates differed from each other only for a total of 367 SNPs, whereas a total of 59,452 SNPs were observed between the isolates and the best matching reference genome (AX1) (Figure 1). The susceptibility phenotype of each of the eight isolates was assessed by spot-test against the phage cocktail APC 2.1 and expressed as “S” when susceptible or “R” when resistant (e.g., “is2S” means that isolate number 2 was susceptible to the phage cocktail) (Table 2 and Supplementary Methods). Among these eight isolates, analysis based on variant calling furthermore revealed four closely related but clearly delineated strains, i.e., clones that differ genetically from other clones, with different phage susceptibility phenotype (Table 2 and Figure 1): Strain 1 (Str1), contained isolates 1 and 3 (is1S and is3R), found in BAL#3 (is1S-Str1/BAL#3) and BAL#5 (is3R-Str1/BAL#5), respectively. Strain 2 (Str2) contained isolate 2 (is2S) from BAL#5 (is2S-Str2/BAL#5). Strain 3 (Str3) contained isolates 5, 6, and 9 (is5S, is6S and is9S), which were found in BAL#6 (is5S-Str3/BAL#6, is6S-Str3/BAL#6) and BAL#9 (is9S-Str3/BAL#9). Strain 4 contained isolates 7 and 8 (is7R and is8R), found in BAL#9 (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) (Table 2). Isolate is1S-Str1/BAL#3 was obtained on 25 July 2017, i.e., 45 days before the first phage therapy session (8 September 2017) and was 100% identical to is3R-Str1/BAL#5 isolated seven days (15 September 2017) after the first phage therapy session (Figure 1). Since both isolates is1S-Str1/BAL#3 and is3R-Str1/BAL#5 are 100% identical, this means that the phage resistance mechanism of isolate is3R-Str1/BAL#5 is not genetically supported (see discussion). Isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6, both phage susceptible, were obtained one month later (12 October 2017) and belonged to still another strain (Strain 3). Finally, on 8 February 2018, i.e., 16 days after the second phage therapy session (23 January 2018), an additional three isolates were obtained from BAL#9. Of these, isolate is9S-Str3/BAL#9 belonged to the same Strain 3 as isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6 and was phage susceptible as well, but isolates 7R and 8R belonged to still another strain (Strain 4) and were both resistant to the phage cocktail. Interestingly, the phage resistant Strain 4 isolates (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) contained mutations compared to the other six isolates that could explain the phage resistance observed for these two isolates (Figure 1): when Strain 1 was used as a reference, a C to A transition at position 1803 of the colicin I receptor gene caused a Tyr601->Stop transition. This receptor has been recognized as a phage receptor [7]. We further studied the course of infection by extracting DNA from the three available A. xylosoxidans-positive BAL samples (BAL#5, #6 and #9), and by quantifying DNA of the different strains by means of strain-specific qPCRs (Table 3 and Supplementary Methods) that had been developed on the basis of the obtained WGS data. qPCRs for Strain 2 and Strain 4 were only positive for the BALs from which these isolates had been cultured, respectively, BAL#5 and BAL#9. qPCR for Strain 1 was only positive for BAL#9, but not for BAL#5 from which isolate 3R had been cultured. qPCR for Strain 3 was positive for all three BALs, although no isolate belonging to Strain 3 was cultured from BAL#5. 3. Discussion and Conclusions We report the first case of decolonization of pandrug-resistant Achromobacter xylosoxidans after a long-term follow-up in a lung-transplanted patient receiving phage therapy. The first actual demonstration and usage of bacteriophages came almost simultaneously from two microbiologists in 1915 (Frederick Twort) and 1917 (Félix d’Hérelle) [8]. After extensive clinical use during the first half of the 20th century, their use has been progressively reduced, mainly due to the discovery, rapid spread, and ease of use of antibiotics for the treatment of infectious diseases [9]. However, bacteriophages gained a new surge of attention, due to the emergence and spread of pandrug-resistant bacteria [8]. Despite decades of clinical experience with bacteriophages in Eastern Europe, very few data are available regarding their clinical efficacy; besides, case-series and few randomized controlled trials (RCT) [10,11,12]. For instance, a RCT assessed the clinical outcome of patients receiving local application of a phage cocktail for the treatment of burn wound infection. This RCT is frequently cited to have shown no clinical benefits, as compared to standard of care. However, the phage titer that was applied (100 pfu/mL) [10], was so extremely low that it precludes to draw any conclusion from this study regarding the usefulness of phage therapy. The limited number of randomized clinical trials has been recently reviewed [13]. Phage therapy has also been used locally, e.g., for the treatment of bone and joint infections [14,15] or intravenously for the treatment of bloodstream infections [16,17]. Considering the high frequency of colonization and infection caused by antibiotic-resistant bacteria among cystic fibrosis and lung-transplanted patients, such as in our patient, the use of bacteriophages appears to be an appealing option in this population [18,19,20]. In the specific case of A. xylosoxidans, thus far a single case-report described the favorable outcome after the use of bacteriophages in a cystic fibrosis patient [21]. Our case-report highlights the many difficulties that need to be taken into account before considering the use of bacteriophages in a clinical setting. These include amongst others an important delay from clinical decision to regulatory agency authorization. Most problematic is the difficult interpretation of the clinical outcome after bacteriophage therapy, considering the long-term persisting airway colonization and the possible evolution of different A. xylosoxidans strains. The second therapeutic attempt relied on a massive lung instillation (injection of the bacteriophage solution in the patient’s lung during fibroscopy) followed by a two-week course of phage nebulization. We used a vibrating mesh nebulizer for phage delivery as this type of nebulizer has been shown to result in a lower phage titer reduction, as compared to jet nebulizers [22]. BAL#9, performed two weeks after the second round of phage therapy (8 February 2018) still grew 105 CFU/mL of A. xylosoxidans, represented by Strains 3 and 4 (according to culture) and by at least Strains 1, 3 and 4 (according to qPCR) (Table 1, Table 2 and Table 3). Sputum cultures remained positive for 18 months, but with only 103 CFU A. xylosoxidans/mL, suggesting a possible upper airway colonization, especially since no A. xylosoxidans could be isolated from BALs sampled in August 2019, and April 2020. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. To more precisely understand the discrepant outcome of our patient, another option would have been to search for active phages from the BAL samples throughout the study, but this was not performed. Despite the observed differences of a total of 367 SNPs between the four strains of eight A. xylosoxidans isolates from the patient, their overall similarity suggests that they originate from a single strain that colonized the patient (who was colonized with A. xylosoxidans years before the start of the phage therapy). Therefore, it is possible that some variants were present already before lung transplantation and before the start of phage therapy. Although it is not possible to exclude the simultaneous presence of these different strains on the basis of our culture results, because only one or two colonies were picked from each BAL culture, analysis by means of four strain-specific qPCR assays of the three BAL samples that were available for study, indicated that Strains 2 and 4 were probably prevalent at a particular moment of the course of infection, whereas Strains 1 and 3 could be identified during the full course of the infection. Phage resistant Strain 4 may have been selected during phage therapy. Phage training has been proposed to overcome phage resistance and increase infectivity against a given bacterial population [23]. However, training phages may take weeks, and therefore might be restricted to the treatment of chronic infections. Regarding more acute infections, the establishment of phage collections (such as the DSMZ, which provided bacteriophages for our patient) is of critical importance to provide phages active against a broad spectrum of bacterial isolates. An interesting point is the observation of two isolates from the same strain (is1S-Str1/BAL#3 and is3R-Str1/BAL#5) that differ regarding their phage susceptibility phenotype despite the absence of genetic support (identical genome). As observed with bacteria where emerging data point out that a resistance phenotype against a given antimicrobial may not be supported by a genotypic substratum [24,25], phage resistance without any change in genotype has also been documented [26]. These genotype/phenotype discrepancies are hypothesized to be possible through epigenetic regulation mechanisms, of which post-transcriptional regulation, which may influence phage receptor expression on the cell surface or phage receptor masking (through capsule synthesis). Further studies are required to better understand the dynamics of in vivo selection of phage-resistant bacteria. Because the patient’s medical condition improved anyhow, a few weeks after the second round of phage instillation, it is possible that phage therapy selected the phage-resistant A. xylosoxidans Strain 4, but that the mutation that caused phage resistance at the same time decreased the virulence of the strain as already described with other bacteria and phages [27,28,29]. Indeed, we observed several mutations which could explain the phage resistance of Strain 4, of which one was especially interesting, since it caused the gain of a stop codon in the colicin I receptor, which has been recognized as a phage receptor in other species [7]. Because in E. coli, the colicin I receptor plays a crucial role in iron transport across the outer membrane [7], loss of this function due to the observed mutation may have caused reduced fitness of the A. xylosoxidans mutant Strain 4. However, from a clinical point-of-view, one cannot exclude a spontaneous improvement, independently of phage therapy and despite months of fruitless antibiotic treatment, highlighting the dire need of randomized clinical trials in order to more convincingly assess the clinical efficacy of bacteriophages. In conclusion, we describe the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. Despite initial persisting airway colonization, the final clinical and microbiological outcome was favorable. Whole genome sequencing of eight A. xylosoxidans isolates and strain-specific qPCR experiments performed on BALs sampled before and after phage therapy allowed to more precisely study the dynamics of lung colonization by different isolates, susceptible or resistant to the applied phage cocktails. Acknowledgments The authors would like to thank L. Lécuyer and V. Boussaud (Unité de Transplantation, HEGP), B. Sabatier, V. Savoldelli, T. Caruba and P. Prognon (Pharmacie HEGP), P. Odou, C. Nassar, C. Berneron and M. Roche (Pharmacie Lille), and E. Deberranger (Hématologie Pédiatrique Lille), for their help in the management of the patient. Microbiological analysis was performed with a funding by the Association Robert Debré pour la Recherche Médicale. The authors would also like to thank Caroline Semaille and Nathalie Morgensztejn from the Agence Nationale de Sécurité du Médicament (ANSM) for their regulatory support. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Materials The following are available online at https://www.mdpi.com/1999-4915/13/1/60/s1, Figure S1: Antibiotic susceptibility testing (AST) of a pandrug-resistant Achromobacter xylosoxidans isolated during a bronchoalveolar lavage of a 12-year-old boy after double lung transplantation, Table S1: Variant calling for the eight Achromobacter xylosoxidans whole genome sequences, see excel file attached, named: “Supplementary Table S1. Variant calling.xls”. Click here for additional data file. Author Contributions D.L. (David Lebeaux) and M.V. conceptualized the study, validated the data, carried out formal analysis, prepared the initial draft, took care of project administration and supervised the study. Data were visualized by H.D. and Y.G. Software analysis was carried out by H.D., S.V., Y.G. D.L. (David Lebeaux), M.M., R.G., I.P., F.C., N.K., J.W., C.R., E.C., D.L. (Damien Lanoy), L.V.S., H.D., J.-P.P., N.D., S.V., Y.G., F.V.N., and M.V. contributed to basic methodology. Data curation was done by D.L. (David Lebeaux), M.M., C.T., S.V., Y.G., and M.V. Funding was acquired by D.L. (David Lebeaux), J.-L.M., F.V.N., and M.V. All authors took part in the investigation and in final reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement The study did not require ethical approval. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Phylogenetic tree built with Whole genome sequences of eight Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. The distance between samples reflects the number of different Single Nucleotide Polymorphisms (SNPs). Each A. xylosoxidans is named as follows: isolate number (is1 to is8) S (or R to depict susceptibility or resistance to phage cocktail)-Strain number/Number of BAL where it has been identified. viruses-13-00060-t001_Table 1Table 1 Results of bronchoalveolar lavages (BALs) and sputum analysis from May 2017, to April 2020, in 12-year-old lung transplanted patient who received two rounds of phage therapy for pandrug-resistant Achromobacter xylosoxidans colonization and infection. Date 19 May 2017 23 May 2017 6 June 2017 12 June 2017 25 July 2017 5 September 2017 8 September 2017 15 September 2017 12 October 2017 27 October 2017 23 January 2018 8 February 2018 5 March 2018 18 May 2018 19 February 2019 19 June 2019 27 August 2019 30 April 2020 Phage therapy ROUND 1 ROUND 2 Tigecycline from 29 June to 31 July Imipenem From 31 July 2017, to 16 February 2018 Number of days, relative to the first round of phage therapy −112 −108 −94 −88 −45 −3 +7 +37 +49 +137 +153 +178 +252 +529 +649 +718 +963 BAL BAL Number * #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 Leukocytes (/mm3) >1000 800 >1000 500 >1000 60 >1000 810 >1000 NA NA NA PMN cells (%) 90 88 90 91 43 30 80 69 82 NA NA NA Quantification of A. x culture (CFU/mL) 104 103 104 103 104 103 104 106 105 104 0 0 Other bacteria (CFU/mL) None None None None None None None None None None 104 S. a 106 S. a Sputum Leucocytes (/field) >25 >25 >25 >25 NA NA NA Quantification of A. x culture (CFU/mL) 105 105 105 106 103 103 103 Other bacteria (CFU/mL) None None None None 108 M. c None 106 S. a A. x: Achromobacter xylosoxidans; M. c: Moraxella catarrhalis; NA: non available; PMN: Polymorphonuclear; RBC: red blood cells; S. a: Staphylococcus aureus. : BAL number in bold indicates BALs for which qPCR had been carried out. viruses-13-00060-t002_Table 2Table 2 Eight Achromobacter xylosoxidans isolates for which whole genome sequencing was carried out. These were isolated from bronchoalveolar lavages in a 12-year-old boy after double lung transplantation. The patient received a first round of phage administration on 8 September 2017. A second round of phage treatment was performed on 23 January 2018. Complete Name Isolate Strain Source of Sampling Date of Sampling Whole Genome Sequence is1S-Str1/BAL#3 is1S Str1 BAL#3 25 July 2017 ERS5044236-UGAX1 is2S-Str2/BAL#5 is2S Str2 BAL#5 15 September 2017 ERS5044237-UGAX2 is3R-Str1/BAL#5 is3R Str1 BAL#5 15 September 2017 ERS5044238-UGAX3 is5S-Str3/BAL#6 is5S Str3 BAL#6 12 October 2017 ERS5044239-UGAX5 is6S-Str3/BAL#6 is6S Str3 BAL#6 12 October 2017 ERS5044240-UGAX6 is7R-Str4/BAL#9 is7R Str4 BAL#9 8 February 2018 ERS5044241-UGAX7 is8R-Str4/BAL#9 is8R Str4 BAL#9 8 February 2018 ERS5044242-UGAX8 is9S-Str3/BAL#9 is9S Str3 BAL#9 8 February 2018 ERS5044243-UGAX9 * Submitted at ENA, 1 September 2020. Study ID: PRJEB39103. BAL: Bronchoalveolar lavage, S: Phage susceptible, R: phage resistant. viruses-13-00060-t003_Table 3Table 3 Results of bacterial culture (grey-shading) and qPCR of three broncho-alveolar lavages for four different Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. Bronchoalveolar Lavage BAL#5 BAL#6 BAL#9 15 September 2017 12 October 2017 8 February 2018 qPCR for Strain Isolates Str1 is1S, is3R N NS P (34.7) Str2 is2S P (36.6) N N Str3 is5S, is6S, is9S P (32.7) P (28.9) P (27.7) Str4 is7R, is8R NS NS P (35.2) Grey-shaded box = isolate cultured from this BAL; N: negative (Cq-value > 40); NS: Nonspecific amplification (melting peak different from that of specific amplification); P: positive qPCR (Cq-value).	MYCOPHENOLATE MOFETIL, POSACONAZOLE, PREDNISOLONE, SULFAMETHOXAZOLE\TRIMETHOPRIM, TACROLIMUS, VALACYCLOVIR HYDROCHLORIDE	DrugsGivenReaction	CC BY	33466377	19,957,163	2021-01-05
What is the weight of the patient?	A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient. Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains. 1. Case Presentation We report the case of a 12-year-old boy (35.5 kg), who received double lung transplantation in 25 March 2017, (at the age of 12) because of cystic fibrosis. Before transplantation, he was colonized with Aspergillus fumigatus, Achromobacter xylosoxidans (first colonization identified in 2013), and Pseudomonas aeruginosa. After transplantation, he experienced acute kidney injury requiring temporary haemodialysis, pulmonary embolism, and persisting airway colonization with A. fumigatus. He was discharged on 29 April 2017, with satisfactory respiratory status; he initially required no home oxygen therapy. His immunosuppressive regimen included prednisolone (25 mg/day), tacrolimus (1.5 mg bis in die (b.i.d.) with a target trough concentration of 6–8 ng/mL), mycophenolate mofetil (1 g b.i.d.) and his antimicrobial prophylaxis was cotrimoxazole (400/80 mg, three times a week), posaconazole (150 mg, oral suspension, ter in die (t.i.d.)), valacyclovir (500 mg b.i.d.), and intravenous immunoglobulins (15 g each three weeks). Between May and June 2017, he experienced progressive shortness of breath, cough and increased sputum production; he subsequently required the addition of 1–1.5 L/min oxygen therapy. Because of a stenosis of the surgical anastomosis of the main left bronchus, a dilatation during rigid bronchoscopy was performed on 19 May and a Montgomery stent was inserted on 2 June. Later on, a stenosis of the surgical anastomosis on the right bronchus intermedius required a dilatation during rigid bronchoscopy followed by the insertion of an Oki stent on 29 June. Several bronchoalveolar lavages (BAL), performed between May and June 2017, revealed inflammation (between 800 and >1000 leukocytes/mm3 with 88–90% of polymorphonuclear cells) and repeatedly grew pandrug-resistant A. xylosoxidans (Table 1 and Supplementary Figure S1) [1]. Lung biopsy performed on 19 June was consistent with lung infection: diffuse acute bronchiolitis with extension to peri-bronchiolar alveoli. Microbiological samples obtained from lung biopsies were negative (bacterial, mycological, and mycobacteriological cultures). The first antibiotic treatment consisted of intravenous tigecycline 50 mg b.i.d. from 29 June to 31 July. As BAL remained positive for A. xylosoxidans and the patient still required oxygen therapy; antibiotic treatment was subsequently switched to imipenem (1200 mg, t.i.d., ~100 mg/Kg/d) on 31 July 2017. Subsequent respiratory samples still grew A. xylosoxidans and the respiratory status did not improve (requiring oxygen at home, 1 L/min). Furthermore, despite antibiotic treatment and correct drainage of the right superior lobe, lung consolidation and micronodules remained. Considering the failure of antibiotic treatment, phage therapy was proposed to the patient and his family, after multidisciplinary discussion. A first cocktail (APC 1.1) containing three lytic phages (JWDelta, JWT and 2-1) active against A. xylosoxidans isolate is1S (designated such as later), selected from the DSMZ collection (Braunschweig, Germany) [2,3], was prepared at the Laboratory for Bacteriology Research (Ghent University, Belgium) and the Laboratory for Molecular and Cellular Technology (Queen Astrid Military Hospital, Brussels, Belgium) using the above-mentioned bacterial isolate, as previously described [4]. The production was completed on 23 August with a final phage titer of 4 × 1010 plaque forming units (pfu)/mL defined by double-agar overlay method [4], an endotoxin level of 4000 EU/mL (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, GenScript, Piscataway Township, NJ, USA) and a pH of 7.3 (in sterile PBS). Regulatory permission for phage importation was obtained on 1 September 2017, from the Agence Nationale de Sécurité du Médicament (ANSM). A first round of phage administration was performed, consisting of 3 nebulizations/day of 5 mL of the filter-sterilized (Sartorius 0.2 µm) phage solution, tenfold diluted in sterile saline on 8 and 9 September, using a vibrating mesh nebulizer (eFLOW rapid, PARI, PARI GmbH, Germany) with a mouth piece. Immediate tolerance was perfect, but no clinical improvement was noted and culture of subsequent respiratory samples remained positive for A. xylosoxidans (104 CFU/mL in the BAL of 15 September). A second cocktail (APC 2.1) was produced, in which phage JWalpha was added to the three lytic phages of cocktail APC 1.1, to improve the therapeutic efficacy. The production was completed on 22 December with a final phage titer of 5 × 109 pfu/mL, an endotoxin level of 1760 EU/mL and a pH of 6.95 (in sterile PBS). Regulatory permission was obtained for phage importation on 16 January 2018 (ANSM). On 23 January, during therapeutic bronchoscopy under general anesthesia, 30 mL of APC2.1, tenfold diluted (in sterile saline) and filter-sterilized, was instilled in each pulmonary lobe through the fibroscope. Immediate tolerance was perfect and the patient was discharged on 24 January with continued phage nebulization at home: three times a day 5 mL of preparation A using a vibrating mesh nebulizer (eFLOW rapid, PARI) with a mouth piece for 14 days until 6 February. Clinical tolerance was perfect again, but the initial clinical status remained unchanged. BAL performed on 8 February 2018, two days after stopping the phage treatment, still grew 105 cfu/mL of A. xylosoxidans. Subsequently, the patient’s respiratory condition slowly improved and oxygen therapy was stopped on 15 February 2018. No clinical worsening was observed after imipenem interruption on 16 February 2018. Sputum culture remained positive for A. xylosoxidans but with a low bacterial density (103 CFU/mL in March and May 2018, and June 2019) and no A. xylosoxidans could be isolated from BALs#10 and #11, sampled on August 2019, and April 2020, respectively (Table 1). The last pulmonary function tests, performed on 17 October 2019, showed best results since lung transplantation, with an FEV1 of 79% and a forced vital capacity of 85%. 2. Microbiological Analysis of Eight A. xylosoxidans Isolates As we observed discrepant outcome in our patient (clinical improvement on the one hand but persisting low-grade airway colonization on the other hand), we decided to study the eight available A. xylosoxidans isolates (is1 to is9) from the patient’s colonization obtained by random picking from the culture plates from 4 distinct BALs (Table 2). These eight isolates, taken before, during and after the phage therapy period (Table 2), could not be differentiated from each other by means of MALDI-TOF [5] or McRAPD typing [6] and all belonged to the epidemic clone, already described previously among Belgian CF patients [5]. Whole genome sequencing (WGS) and de novo genome assembly for these eight isolates was carried out (Supplementary Methods and Table S1). The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Total genome length for all eight isolates was 6.44–6.50 Mbp, with a GC content of 67.5%. All eight isolates showed most similarity with reference genome GCF_008432465 (AX1), which corresponds to the type strain of A. xylosoxidans subsp. xylosoxidans (ATCC 27061T, CCUG 12689T, DSMZ 10346T, LMG 1863T, and NCTC 10807T), i.e., between 88.38 and 89.60% on the basis of mapped sequence reads, and between 98.71% and 98.76% on the basis of average nucleotide identity (ANI). Indeed, the overall similarity between the genome sequences of the eight isolates was high, i.e., 99.60%, as assessed by digital DNA hybridization, and more than 99.97% according to ANI-analysis. This was confirmed by variant calling where we observed that the isolates differed from each other only for a total of 367 SNPs, whereas a total of 59,452 SNPs were observed between the isolates and the best matching reference genome (AX1) (Figure 1). The susceptibility phenotype of each of the eight isolates was assessed by spot-test against the phage cocktail APC 2.1 and expressed as “S” when susceptible or “R” when resistant (e.g., “is2S” means that isolate number 2 was susceptible to the phage cocktail) (Table 2 and Supplementary Methods). Among these eight isolates, analysis based on variant calling furthermore revealed four closely related but clearly delineated strains, i.e., clones that differ genetically from other clones, with different phage susceptibility phenotype (Table 2 and Figure 1): Strain 1 (Str1), contained isolates 1 and 3 (is1S and is3R), found in BAL#3 (is1S-Str1/BAL#3) and BAL#5 (is3R-Str1/BAL#5), respectively. Strain 2 (Str2) contained isolate 2 (is2S) from BAL#5 (is2S-Str2/BAL#5). Strain 3 (Str3) contained isolates 5, 6, and 9 (is5S, is6S and is9S), which were found in BAL#6 (is5S-Str3/BAL#6, is6S-Str3/BAL#6) and BAL#9 (is9S-Str3/BAL#9). Strain 4 contained isolates 7 and 8 (is7R and is8R), found in BAL#9 (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) (Table 2). Isolate is1S-Str1/BAL#3 was obtained on 25 July 2017, i.e., 45 days before the first phage therapy session (8 September 2017) and was 100% identical to is3R-Str1/BAL#5 isolated seven days (15 September 2017) after the first phage therapy session (Figure 1). Since both isolates is1S-Str1/BAL#3 and is3R-Str1/BAL#5 are 100% identical, this means that the phage resistance mechanism of isolate is3R-Str1/BAL#5 is not genetically supported (see discussion). Isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6, both phage susceptible, were obtained one month later (12 October 2017) and belonged to still another strain (Strain 3). Finally, on 8 February 2018, i.e., 16 days after the second phage therapy session (23 January 2018), an additional three isolates were obtained from BAL#9. Of these, isolate is9S-Str3/BAL#9 belonged to the same Strain 3 as isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6 and was phage susceptible as well, but isolates 7R and 8R belonged to still another strain (Strain 4) and were both resistant to the phage cocktail. Interestingly, the phage resistant Strain 4 isolates (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) contained mutations compared to the other six isolates that could explain the phage resistance observed for these two isolates (Figure 1): when Strain 1 was used as a reference, a C to A transition at position 1803 of the colicin I receptor gene caused a Tyr601->Stop transition. This receptor has been recognized as a phage receptor [7]. We further studied the course of infection by extracting DNA from the three available A. xylosoxidans-positive BAL samples (BAL#5, #6 and #9), and by quantifying DNA of the different strains by means of strain-specific qPCRs (Table 3 and Supplementary Methods) that had been developed on the basis of the obtained WGS data. qPCRs for Strain 2 and Strain 4 were only positive for the BALs from which these isolates had been cultured, respectively, BAL#5 and BAL#9. qPCR for Strain 1 was only positive for BAL#9, but not for BAL#5 from which isolate 3R had been cultured. qPCR for Strain 3 was positive for all three BALs, although no isolate belonging to Strain 3 was cultured from BAL#5. 3. Discussion and Conclusions We report the first case of decolonization of pandrug-resistant Achromobacter xylosoxidans after a long-term follow-up in a lung-transplanted patient receiving phage therapy. The first actual demonstration and usage of bacteriophages came almost simultaneously from two microbiologists in 1915 (Frederick Twort) and 1917 (Félix d’Hérelle) [8]. After extensive clinical use during the first half of the 20th century, their use has been progressively reduced, mainly due to the discovery, rapid spread, and ease of use of antibiotics for the treatment of infectious diseases [9]. However, bacteriophages gained a new surge of attention, due to the emergence and spread of pandrug-resistant bacteria [8]. Despite decades of clinical experience with bacteriophages in Eastern Europe, very few data are available regarding their clinical efficacy; besides, case-series and few randomized controlled trials (RCT) [10,11,12]. For instance, a RCT assessed the clinical outcome of patients receiving local application of a phage cocktail for the treatment of burn wound infection. This RCT is frequently cited to have shown no clinical benefits, as compared to standard of care. However, the phage titer that was applied (100 pfu/mL) [10], was so extremely low that it precludes to draw any conclusion from this study regarding the usefulness of phage therapy. The limited number of randomized clinical trials has been recently reviewed [13]. Phage therapy has also been used locally, e.g., for the treatment of bone and joint infections [14,15] or intravenously for the treatment of bloodstream infections [16,17]. Considering the high frequency of colonization and infection caused by antibiotic-resistant bacteria among cystic fibrosis and lung-transplanted patients, such as in our patient, the use of bacteriophages appears to be an appealing option in this population [18,19,20]. In the specific case of A. xylosoxidans, thus far a single case-report described the favorable outcome after the use of bacteriophages in a cystic fibrosis patient [21]. Our case-report highlights the many difficulties that need to be taken into account before considering the use of bacteriophages in a clinical setting. These include amongst others an important delay from clinical decision to regulatory agency authorization. Most problematic is the difficult interpretation of the clinical outcome after bacteriophage therapy, considering the long-term persisting airway colonization and the possible evolution of different A. xylosoxidans strains. The second therapeutic attempt relied on a massive lung instillation (injection of the bacteriophage solution in the patient’s lung during fibroscopy) followed by a two-week course of phage nebulization. We used a vibrating mesh nebulizer for phage delivery as this type of nebulizer has been shown to result in a lower phage titer reduction, as compared to jet nebulizers [22]. BAL#9, performed two weeks after the second round of phage therapy (8 February 2018) still grew 105 CFU/mL of A. xylosoxidans, represented by Strains 3 and 4 (according to culture) and by at least Strains 1, 3 and 4 (according to qPCR) (Table 1, Table 2 and Table 3). Sputum cultures remained positive for 18 months, but with only 103 CFU A. xylosoxidans/mL, suggesting a possible upper airway colonization, especially since no A. xylosoxidans could be isolated from BALs sampled in August 2019, and April 2020. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. To more precisely understand the discrepant outcome of our patient, another option would have been to search for active phages from the BAL samples throughout the study, but this was not performed. Despite the observed differences of a total of 367 SNPs between the four strains of eight A. xylosoxidans isolates from the patient, their overall similarity suggests that they originate from a single strain that colonized the patient (who was colonized with A. xylosoxidans years before the start of the phage therapy). Therefore, it is possible that some variants were present already before lung transplantation and before the start of phage therapy. Although it is not possible to exclude the simultaneous presence of these different strains on the basis of our culture results, because only one or two colonies were picked from each BAL culture, analysis by means of four strain-specific qPCR assays of the three BAL samples that were available for study, indicated that Strains 2 and 4 were probably prevalent at a particular moment of the course of infection, whereas Strains 1 and 3 could be identified during the full course of the infection. Phage resistant Strain 4 may have been selected during phage therapy. Phage training has been proposed to overcome phage resistance and increase infectivity against a given bacterial population [23]. However, training phages may take weeks, and therefore might be restricted to the treatment of chronic infections. Regarding more acute infections, the establishment of phage collections (such as the DSMZ, which provided bacteriophages for our patient) is of critical importance to provide phages active against a broad spectrum of bacterial isolates. An interesting point is the observation of two isolates from the same strain (is1S-Str1/BAL#3 and is3R-Str1/BAL#5) that differ regarding their phage susceptibility phenotype despite the absence of genetic support (identical genome). As observed with bacteria where emerging data point out that a resistance phenotype against a given antimicrobial may not be supported by a genotypic substratum [24,25], phage resistance without any change in genotype has also been documented [26]. These genotype/phenotype discrepancies are hypothesized to be possible through epigenetic regulation mechanisms, of which post-transcriptional regulation, which may influence phage receptor expression on the cell surface or phage receptor masking (through capsule synthesis). Further studies are required to better understand the dynamics of in vivo selection of phage-resistant bacteria. Because the patient’s medical condition improved anyhow, a few weeks after the second round of phage instillation, it is possible that phage therapy selected the phage-resistant A. xylosoxidans Strain 4, but that the mutation that caused phage resistance at the same time decreased the virulence of the strain as already described with other bacteria and phages [27,28,29]. Indeed, we observed several mutations which could explain the phage resistance of Strain 4, of which one was especially interesting, since it caused the gain of a stop codon in the colicin I receptor, which has been recognized as a phage receptor in other species [7]. Because in E. coli, the colicin I receptor plays a crucial role in iron transport across the outer membrane [7], loss of this function due to the observed mutation may have caused reduced fitness of the A. xylosoxidans mutant Strain 4. However, from a clinical point-of-view, one cannot exclude a spontaneous improvement, independently of phage therapy and despite months of fruitless antibiotic treatment, highlighting the dire need of randomized clinical trials in order to more convincingly assess the clinical efficacy of bacteriophages. In conclusion, we describe the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. Despite initial persisting airway colonization, the final clinical and microbiological outcome was favorable. Whole genome sequencing of eight A. xylosoxidans isolates and strain-specific qPCR experiments performed on BALs sampled before and after phage therapy allowed to more precisely study the dynamics of lung colonization by different isolates, susceptible or resistant to the applied phage cocktails. Acknowledgments The authors would like to thank L. Lécuyer and V. Boussaud (Unité de Transplantation, HEGP), B. Sabatier, V. Savoldelli, T. Caruba and P. Prognon (Pharmacie HEGP), P. Odou, C. Nassar, C. Berneron and M. Roche (Pharmacie Lille), and E. Deberranger (Hématologie Pédiatrique Lille), for their help in the management of the patient. Microbiological analysis was performed with a funding by the Association Robert Debré pour la Recherche Médicale. The authors would also like to thank Caroline Semaille and Nathalie Morgensztejn from the Agence Nationale de Sécurité du Médicament (ANSM) for their regulatory support. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Materials The following are available online at https://www.mdpi.com/1999-4915/13/1/60/s1, Figure S1: Antibiotic susceptibility testing (AST) of a pandrug-resistant Achromobacter xylosoxidans isolated during a bronchoalveolar lavage of a 12-year-old boy after double lung transplantation, Table S1: Variant calling for the eight Achromobacter xylosoxidans whole genome sequences, see excel file attached, named: “Supplementary Table S1. Variant calling.xls”. Click here for additional data file. Author Contributions D.L. (David Lebeaux) and M.V. conceptualized the study, validated the data, carried out formal analysis, prepared the initial draft, took care of project administration and supervised the study. Data were visualized by H.D. and Y.G. Software analysis was carried out by H.D., S.V., Y.G. D.L. (David Lebeaux), M.M., R.G., I.P., F.C., N.K., J.W., C.R., E.C., D.L. (Damien Lanoy), L.V.S., H.D., J.-P.P., N.D., S.V., Y.G., F.V.N., and M.V. contributed to basic methodology. Data curation was done by D.L. (David Lebeaux), M.M., C.T., S.V., Y.G., and M.V. Funding was acquired by D.L. (David Lebeaux), J.-L.M., F.V.N., and M.V. All authors took part in the investigation and in final reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement The study did not require ethical approval. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Phylogenetic tree built with Whole genome sequences of eight Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. The distance between samples reflects the number of different Single Nucleotide Polymorphisms (SNPs). Each A. xylosoxidans is named as follows: isolate number (is1 to is8) S (or R to depict susceptibility or resistance to phage cocktail)-Strain number/Number of BAL where it has been identified. viruses-13-00060-t001_Table 1Table 1 Results of bronchoalveolar lavages (BALs) and sputum analysis from May 2017, to April 2020, in 12-year-old lung transplanted patient who received two rounds of phage therapy for pandrug-resistant Achromobacter xylosoxidans colonization and infection. Date 19 May 2017 23 May 2017 6 June 2017 12 June 2017 25 July 2017 5 September 2017 8 September 2017 15 September 2017 12 October 2017 27 October 2017 23 January 2018 8 February 2018 5 March 2018 18 May 2018 19 February 2019 19 June 2019 27 August 2019 30 April 2020 Phage therapy ROUND 1 ROUND 2 Tigecycline from 29 June to 31 July Imipenem From 31 July 2017, to 16 February 2018 Number of days, relative to the first round of phage therapy −112 −108 −94 −88 −45 −3 +7 +37 +49 +137 +153 +178 +252 +529 +649 +718 +963 BAL BAL Number * #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 Leukocytes (/mm3) >1000 800 >1000 500 >1000 60 >1000 810 >1000 NA NA NA PMN cells (%) 90 88 90 91 43 30 80 69 82 NA NA NA Quantification of A. x culture (CFU/mL) 104 103 104 103 104 103 104 106 105 104 0 0 Other bacteria (CFU/mL) None None None None None None None None None None 104 S. a 106 S. a Sputum Leucocytes (/field) >25 >25 >25 >25 NA NA NA Quantification of A. x culture (CFU/mL) 105 105 105 106 103 103 103 Other bacteria (CFU/mL) None None None None 108 M. c None 106 S. a A. x: Achromobacter xylosoxidans; M. c: Moraxella catarrhalis; NA: non available; PMN: Polymorphonuclear; RBC: red blood cells; S. a: Staphylococcus aureus. : BAL number in bold indicates BALs for which qPCR had been carried out. viruses-13-00060-t002_Table 2Table 2 Eight Achromobacter xylosoxidans isolates for which whole genome sequencing was carried out. These were isolated from bronchoalveolar lavages in a 12-year-old boy after double lung transplantation. The patient received a first round of phage administration on 8 September 2017. A second round of phage treatment was performed on 23 January 2018. Complete Name Isolate Strain Source of Sampling Date of Sampling Whole Genome Sequence is1S-Str1/BAL#3 is1S Str1 BAL#3 25 July 2017 ERS5044236-UGAX1 is2S-Str2/BAL#5 is2S Str2 BAL#5 15 September 2017 ERS5044237-UGAX2 is3R-Str1/BAL#5 is3R Str1 BAL#5 15 September 2017 ERS5044238-UGAX3 is5S-Str3/BAL#6 is5S Str3 BAL#6 12 October 2017 ERS5044239-UGAX5 is6S-Str3/BAL#6 is6S Str3 BAL#6 12 October 2017 ERS5044240-UGAX6 is7R-Str4/BAL#9 is7R Str4 BAL#9 8 February 2018 ERS5044241-UGAX7 is8R-Str4/BAL#9 is8R Str4 BAL#9 8 February 2018 ERS5044242-UGAX8 is9S-Str3/BAL#9 is9S Str3 BAL#9 8 February 2018 ERS5044243-UGAX9 * Submitted at ENA, 1 September 2020. Study ID: PRJEB39103. BAL: Bronchoalveolar lavage, S: Phage susceptible, R: phage resistant. viruses-13-00060-t003_Table 3Table 3 Results of bacterial culture (grey-shading) and qPCR of three broncho-alveolar lavages for four different Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. Bronchoalveolar Lavage BAL#5 BAL#6 BAL#9 15 September 2017 12 October 2017 8 February 2018 qPCR for Strain Isolates Str1 is1S, is3R N NS P (34.7) Str2 is2S P (36.6) N N Str3 is5S, is6S, is9S P (32.7) P (28.9) P (27.7) Str4 is7R, is8R NS NS P (35.2) Grey-shaded box = isolate cultured from this BAL; N: negative (Cq-value > 40); NS: Nonspecific amplification (melting peak different from that of specific amplification); P: positive qPCR (Cq-value).	35.5 kg.	Weight	CC BY	33466377	19,957,163	2021-01-05
What was the administration route of drug 'POSACONAZOLE'?	A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient. Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains. 1. Case Presentation We report the case of a 12-year-old boy (35.5 kg), who received double lung transplantation in 25 March 2017, (at the age of 12) because of cystic fibrosis. Before transplantation, he was colonized with Aspergillus fumigatus, Achromobacter xylosoxidans (first colonization identified in 2013), and Pseudomonas aeruginosa. After transplantation, he experienced acute kidney injury requiring temporary haemodialysis, pulmonary embolism, and persisting airway colonization with A. fumigatus. He was discharged on 29 April 2017, with satisfactory respiratory status; he initially required no home oxygen therapy. His immunosuppressive regimen included prednisolone (25 mg/day), tacrolimus (1.5 mg bis in die (b.i.d.) with a target trough concentration of 6–8 ng/mL), mycophenolate mofetil (1 g b.i.d.) and his antimicrobial prophylaxis was cotrimoxazole (400/80 mg, three times a week), posaconazole (150 mg, oral suspension, ter in die (t.i.d.)), valacyclovir (500 mg b.i.d.), and intravenous immunoglobulins (15 g each three weeks). Between May and June 2017, he experienced progressive shortness of breath, cough and increased sputum production; he subsequently required the addition of 1–1.5 L/min oxygen therapy. Because of a stenosis of the surgical anastomosis of the main left bronchus, a dilatation during rigid bronchoscopy was performed on 19 May and a Montgomery stent was inserted on 2 June. Later on, a stenosis of the surgical anastomosis on the right bronchus intermedius required a dilatation during rigid bronchoscopy followed by the insertion of an Oki stent on 29 June. Several bronchoalveolar lavages (BAL), performed between May and June 2017, revealed inflammation (between 800 and >1000 leukocytes/mm3 with 88–90% of polymorphonuclear cells) and repeatedly grew pandrug-resistant A. xylosoxidans (Table 1 and Supplementary Figure S1) [1]. Lung biopsy performed on 19 June was consistent with lung infection: diffuse acute bronchiolitis with extension to peri-bronchiolar alveoli. Microbiological samples obtained from lung biopsies were negative (bacterial, mycological, and mycobacteriological cultures). The first antibiotic treatment consisted of intravenous tigecycline 50 mg b.i.d. from 29 June to 31 July. As BAL remained positive for A. xylosoxidans and the patient still required oxygen therapy; antibiotic treatment was subsequently switched to imipenem (1200 mg, t.i.d., ~100 mg/Kg/d) on 31 July 2017. Subsequent respiratory samples still grew A. xylosoxidans and the respiratory status did not improve (requiring oxygen at home, 1 L/min). Furthermore, despite antibiotic treatment and correct drainage of the right superior lobe, lung consolidation and micronodules remained. Considering the failure of antibiotic treatment, phage therapy was proposed to the patient and his family, after multidisciplinary discussion. A first cocktail (APC 1.1) containing three lytic phages (JWDelta, JWT and 2-1) active against A. xylosoxidans isolate is1S (designated such as later), selected from the DSMZ collection (Braunschweig, Germany) [2,3], was prepared at the Laboratory for Bacteriology Research (Ghent University, Belgium) and the Laboratory for Molecular and Cellular Technology (Queen Astrid Military Hospital, Brussels, Belgium) using the above-mentioned bacterial isolate, as previously described [4]. The production was completed on 23 August with a final phage titer of 4 × 1010 plaque forming units (pfu)/mL defined by double-agar overlay method [4], an endotoxin level of 4000 EU/mL (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, GenScript, Piscataway Township, NJ, USA) and a pH of 7.3 (in sterile PBS). Regulatory permission for phage importation was obtained on 1 September 2017, from the Agence Nationale de Sécurité du Médicament (ANSM). A first round of phage administration was performed, consisting of 3 nebulizations/day of 5 mL of the filter-sterilized (Sartorius 0.2 µm) phage solution, tenfold diluted in sterile saline on 8 and 9 September, using a vibrating mesh nebulizer (eFLOW rapid, PARI, PARI GmbH, Germany) with a mouth piece. Immediate tolerance was perfect, but no clinical improvement was noted and culture of subsequent respiratory samples remained positive for A. xylosoxidans (104 CFU/mL in the BAL of 15 September). A second cocktail (APC 2.1) was produced, in which phage JWalpha was added to the three lytic phages of cocktail APC 1.1, to improve the therapeutic efficacy. The production was completed on 22 December with a final phage titer of 5 × 109 pfu/mL, an endotoxin level of 1760 EU/mL and a pH of 6.95 (in sterile PBS). Regulatory permission was obtained for phage importation on 16 January 2018 (ANSM). On 23 January, during therapeutic bronchoscopy under general anesthesia, 30 mL of APC2.1, tenfold diluted (in sterile saline) and filter-sterilized, was instilled in each pulmonary lobe through the fibroscope. Immediate tolerance was perfect and the patient was discharged on 24 January with continued phage nebulization at home: three times a day 5 mL of preparation A using a vibrating mesh nebulizer (eFLOW rapid, PARI) with a mouth piece for 14 days until 6 February. Clinical tolerance was perfect again, but the initial clinical status remained unchanged. BAL performed on 8 February 2018, two days after stopping the phage treatment, still grew 105 cfu/mL of A. xylosoxidans. Subsequently, the patient’s respiratory condition slowly improved and oxygen therapy was stopped on 15 February 2018. No clinical worsening was observed after imipenem interruption on 16 February 2018. Sputum culture remained positive for A. xylosoxidans but with a low bacterial density (103 CFU/mL in March and May 2018, and June 2019) and no A. xylosoxidans could be isolated from BALs#10 and #11, sampled on August 2019, and April 2020, respectively (Table 1). The last pulmonary function tests, performed on 17 October 2019, showed best results since lung transplantation, with an FEV1 of 79% and a forced vital capacity of 85%. 2. Microbiological Analysis of Eight A. xylosoxidans Isolates As we observed discrepant outcome in our patient (clinical improvement on the one hand but persisting low-grade airway colonization on the other hand), we decided to study the eight available A. xylosoxidans isolates (is1 to is9) from the patient’s colonization obtained by random picking from the culture plates from 4 distinct BALs (Table 2). These eight isolates, taken before, during and after the phage therapy period (Table 2), could not be differentiated from each other by means of MALDI-TOF [5] or McRAPD typing [6] and all belonged to the epidemic clone, already described previously among Belgian CF patients [5]. Whole genome sequencing (WGS) and de novo genome assembly for these eight isolates was carried out (Supplementary Methods and Table S1). The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Total genome length for all eight isolates was 6.44–6.50 Mbp, with a GC content of 67.5%. All eight isolates showed most similarity with reference genome GCF_008432465 (AX1), which corresponds to the type strain of A. xylosoxidans subsp. xylosoxidans (ATCC 27061T, CCUG 12689T, DSMZ 10346T, LMG 1863T, and NCTC 10807T), i.e., between 88.38 and 89.60% on the basis of mapped sequence reads, and between 98.71% and 98.76% on the basis of average nucleotide identity (ANI). Indeed, the overall similarity between the genome sequences of the eight isolates was high, i.e., 99.60%, as assessed by digital DNA hybridization, and more than 99.97% according to ANI-analysis. This was confirmed by variant calling where we observed that the isolates differed from each other only for a total of 367 SNPs, whereas a total of 59,452 SNPs were observed between the isolates and the best matching reference genome (AX1) (Figure 1). The susceptibility phenotype of each of the eight isolates was assessed by spot-test against the phage cocktail APC 2.1 and expressed as “S” when susceptible or “R” when resistant (e.g., “is2S” means that isolate number 2 was susceptible to the phage cocktail) (Table 2 and Supplementary Methods). Among these eight isolates, analysis based on variant calling furthermore revealed four closely related but clearly delineated strains, i.e., clones that differ genetically from other clones, with different phage susceptibility phenotype (Table 2 and Figure 1): Strain 1 (Str1), contained isolates 1 and 3 (is1S and is3R), found in BAL#3 (is1S-Str1/BAL#3) and BAL#5 (is3R-Str1/BAL#5), respectively. Strain 2 (Str2) contained isolate 2 (is2S) from BAL#5 (is2S-Str2/BAL#5). Strain 3 (Str3) contained isolates 5, 6, and 9 (is5S, is6S and is9S), which were found in BAL#6 (is5S-Str3/BAL#6, is6S-Str3/BAL#6) and BAL#9 (is9S-Str3/BAL#9). Strain 4 contained isolates 7 and 8 (is7R and is8R), found in BAL#9 (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) (Table 2). Isolate is1S-Str1/BAL#3 was obtained on 25 July 2017, i.e., 45 days before the first phage therapy session (8 September 2017) and was 100% identical to is3R-Str1/BAL#5 isolated seven days (15 September 2017) after the first phage therapy session (Figure 1). Since both isolates is1S-Str1/BAL#3 and is3R-Str1/BAL#5 are 100% identical, this means that the phage resistance mechanism of isolate is3R-Str1/BAL#5 is not genetically supported (see discussion). Isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6, both phage susceptible, were obtained one month later (12 October 2017) and belonged to still another strain (Strain 3). Finally, on 8 February 2018, i.e., 16 days after the second phage therapy session (23 January 2018), an additional three isolates were obtained from BAL#9. Of these, isolate is9S-Str3/BAL#9 belonged to the same Strain 3 as isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6 and was phage susceptible as well, but isolates 7R and 8R belonged to still another strain (Strain 4) and were both resistant to the phage cocktail. Interestingly, the phage resistant Strain 4 isolates (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) contained mutations compared to the other six isolates that could explain the phage resistance observed for these two isolates (Figure 1): when Strain 1 was used as a reference, a C to A transition at position 1803 of the colicin I receptor gene caused a Tyr601->Stop transition. This receptor has been recognized as a phage receptor [7]. We further studied the course of infection by extracting DNA from the three available A. xylosoxidans-positive BAL samples (BAL#5, #6 and #9), and by quantifying DNA of the different strains by means of strain-specific qPCRs (Table 3 and Supplementary Methods) that had been developed on the basis of the obtained WGS data. qPCRs for Strain 2 and Strain 4 were only positive for the BALs from which these isolates had been cultured, respectively, BAL#5 and BAL#9. qPCR for Strain 1 was only positive for BAL#9, but not for BAL#5 from which isolate 3R had been cultured. qPCR for Strain 3 was positive for all three BALs, although no isolate belonging to Strain 3 was cultured from BAL#5. 3. Discussion and Conclusions We report the first case of decolonization of pandrug-resistant Achromobacter xylosoxidans after a long-term follow-up in a lung-transplanted patient receiving phage therapy. The first actual demonstration and usage of bacteriophages came almost simultaneously from two microbiologists in 1915 (Frederick Twort) and 1917 (Félix d’Hérelle) [8]. After extensive clinical use during the first half of the 20th century, their use has been progressively reduced, mainly due to the discovery, rapid spread, and ease of use of antibiotics for the treatment of infectious diseases [9]. However, bacteriophages gained a new surge of attention, due to the emergence and spread of pandrug-resistant bacteria [8]. Despite decades of clinical experience with bacteriophages in Eastern Europe, very few data are available regarding their clinical efficacy; besides, case-series and few randomized controlled trials (RCT) [10,11,12]. For instance, a RCT assessed the clinical outcome of patients receiving local application of a phage cocktail for the treatment of burn wound infection. This RCT is frequently cited to have shown no clinical benefits, as compared to standard of care. However, the phage titer that was applied (100 pfu/mL) [10], was so extremely low that it precludes to draw any conclusion from this study regarding the usefulness of phage therapy. The limited number of randomized clinical trials has been recently reviewed [13]. Phage therapy has also been used locally, e.g., for the treatment of bone and joint infections [14,15] or intravenously for the treatment of bloodstream infections [16,17]. Considering the high frequency of colonization and infection caused by antibiotic-resistant bacteria among cystic fibrosis and lung-transplanted patients, such as in our patient, the use of bacteriophages appears to be an appealing option in this population [18,19,20]. In the specific case of A. xylosoxidans, thus far a single case-report described the favorable outcome after the use of bacteriophages in a cystic fibrosis patient [21]. Our case-report highlights the many difficulties that need to be taken into account before considering the use of bacteriophages in a clinical setting. These include amongst others an important delay from clinical decision to regulatory agency authorization. Most problematic is the difficult interpretation of the clinical outcome after bacteriophage therapy, considering the long-term persisting airway colonization and the possible evolution of different A. xylosoxidans strains. The second therapeutic attempt relied on a massive lung instillation (injection of the bacteriophage solution in the patient’s lung during fibroscopy) followed by a two-week course of phage nebulization. We used a vibrating mesh nebulizer for phage delivery as this type of nebulizer has been shown to result in a lower phage titer reduction, as compared to jet nebulizers [22]. BAL#9, performed two weeks after the second round of phage therapy (8 February 2018) still grew 105 CFU/mL of A. xylosoxidans, represented by Strains 3 and 4 (according to culture) and by at least Strains 1, 3 and 4 (according to qPCR) (Table 1, Table 2 and Table 3). Sputum cultures remained positive for 18 months, but with only 103 CFU A. xylosoxidans/mL, suggesting a possible upper airway colonization, especially since no A. xylosoxidans could be isolated from BALs sampled in August 2019, and April 2020. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. To more precisely understand the discrepant outcome of our patient, another option would have been to search for active phages from the BAL samples throughout the study, but this was not performed. Despite the observed differences of a total of 367 SNPs between the four strains of eight A. xylosoxidans isolates from the patient, their overall similarity suggests that they originate from a single strain that colonized the patient (who was colonized with A. xylosoxidans years before the start of the phage therapy). Therefore, it is possible that some variants were present already before lung transplantation and before the start of phage therapy. Although it is not possible to exclude the simultaneous presence of these different strains on the basis of our culture results, because only one or two colonies were picked from each BAL culture, analysis by means of four strain-specific qPCR assays of the three BAL samples that were available for study, indicated that Strains 2 and 4 were probably prevalent at a particular moment of the course of infection, whereas Strains 1 and 3 could be identified during the full course of the infection. Phage resistant Strain 4 may have been selected during phage therapy. Phage training has been proposed to overcome phage resistance and increase infectivity against a given bacterial population [23]. However, training phages may take weeks, and therefore might be restricted to the treatment of chronic infections. Regarding more acute infections, the establishment of phage collections (such as the DSMZ, which provided bacteriophages for our patient) is of critical importance to provide phages active against a broad spectrum of bacterial isolates. An interesting point is the observation of two isolates from the same strain (is1S-Str1/BAL#3 and is3R-Str1/BAL#5) that differ regarding their phage susceptibility phenotype despite the absence of genetic support (identical genome). As observed with bacteria where emerging data point out that a resistance phenotype against a given antimicrobial may not be supported by a genotypic substratum [24,25], phage resistance without any change in genotype has also been documented [26]. These genotype/phenotype discrepancies are hypothesized to be possible through epigenetic regulation mechanisms, of which post-transcriptional regulation, which may influence phage receptor expression on the cell surface or phage receptor masking (through capsule synthesis). Further studies are required to better understand the dynamics of in vivo selection of phage-resistant bacteria. Because the patient’s medical condition improved anyhow, a few weeks after the second round of phage instillation, it is possible that phage therapy selected the phage-resistant A. xylosoxidans Strain 4, but that the mutation that caused phage resistance at the same time decreased the virulence of the strain as already described with other bacteria and phages [27,28,29]. Indeed, we observed several mutations which could explain the phage resistance of Strain 4, of which one was especially interesting, since it caused the gain of a stop codon in the colicin I receptor, which has been recognized as a phage receptor in other species [7]. Because in E. coli, the colicin I receptor plays a crucial role in iron transport across the outer membrane [7], loss of this function due to the observed mutation may have caused reduced fitness of the A. xylosoxidans mutant Strain 4. However, from a clinical point-of-view, one cannot exclude a spontaneous improvement, independently of phage therapy and despite months of fruitless antibiotic treatment, highlighting the dire need of randomized clinical trials in order to more convincingly assess the clinical efficacy of bacteriophages. In conclusion, we describe the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. Despite initial persisting airway colonization, the final clinical and microbiological outcome was favorable. Whole genome sequencing of eight A. xylosoxidans isolates and strain-specific qPCR experiments performed on BALs sampled before and after phage therapy allowed to more precisely study the dynamics of lung colonization by different isolates, susceptible or resistant to the applied phage cocktails. Acknowledgments The authors would like to thank L. Lécuyer and V. Boussaud (Unité de Transplantation, HEGP), B. Sabatier, V. Savoldelli, T. Caruba and P. Prognon (Pharmacie HEGP), P. Odou, C. Nassar, C. Berneron and M. Roche (Pharmacie Lille), and E. Deberranger (Hématologie Pédiatrique Lille), for their help in the management of the patient. Microbiological analysis was performed with a funding by the Association Robert Debré pour la Recherche Médicale. The authors would also like to thank Caroline Semaille and Nathalie Morgensztejn from the Agence Nationale de Sécurité du Médicament (ANSM) for their regulatory support. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Materials The following are available online at https://www.mdpi.com/1999-4915/13/1/60/s1, Figure S1: Antibiotic susceptibility testing (AST) of a pandrug-resistant Achromobacter xylosoxidans isolated during a bronchoalveolar lavage of a 12-year-old boy after double lung transplantation, Table S1: Variant calling for the eight Achromobacter xylosoxidans whole genome sequences, see excel file attached, named: “Supplementary Table S1. Variant calling.xls”. Click here for additional data file. Author Contributions D.L. (David Lebeaux) and M.V. conceptualized the study, validated the data, carried out formal analysis, prepared the initial draft, took care of project administration and supervised the study. Data were visualized by H.D. and Y.G. Software analysis was carried out by H.D., S.V., Y.G. D.L. (David Lebeaux), M.M., R.G., I.P., F.C., N.K., J.W., C.R., E.C., D.L. (Damien Lanoy), L.V.S., H.D., J.-P.P., N.D., S.V., Y.G., F.V.N., and M.V. contributed to basic methodology. Data curation was done by D.L. (David Lebeaux), M.M., C.T., S.V., Y.G., and M.V. Funding was acquired by D.L. (David Lebeaux), J.-L.M., F.V.N., and M.V. All authors took part in the investigation and in final reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement The study did not require ethical approval. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Phylogenetic tree built with Whole genome sequences of eight Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. The distance between samples reflects the number of different Single Nucleotide Polymorphisms (SNPs). Each A. xylosoxidans is named as follows: isolate number (is1 to is8) S (or R to depict susceptibility or resistance to phage cocktail)-Strain number/Number of BAL where it has been identified. viruses-13-00060-t001_Table 1Table 1 Results of bronchoalveolar lavages (BALs) and sputum analysis from May 2017, to April 2020, in 12-year-old lung transplanted patient who received two rounds of phage therapy for pandrug-resistant Achromobacter xylosoxidans colonization and infection. Date 19 May 2017 23 May 2017 6 June 2017 12 June 2017 25 July 2017 5 September 2017 8 September 2017 15 September 2017 12 October 2017 27 October 2017 23 January 2018 8 February 2018 5 March 2018 18 May 2018 19 February 2019 19 June 2019 27 August 2019 30 April 2020 Phage therapy ROUND 1 ROUND 2 Tigecycline from 29 June to 31 July Imipenem From 31 July 2017, to 16 February 2018 Number of days, relative to the first round of phage therapy −112 −108 −94 −88 −45 −3 +7 +37 +49 +137 +153 +178 +252 +529 +649 +718 +963 BAL BAL Number * #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 Leukocytes (/mm3) >1000 800 >1000 500 >1000 60 >1000 810 >1000 NA NA NA PMN cells (%) 90 88 90 91 43 30 80 69 82 NA NA NA Quantification of A. x culture (CFU/mL) 104 103 104 103 104 103 104 106 105 104 0 0 Other bacteria (CFU/mL) None None None None None None None None None None 104 S. a 106 S. a Sputum Leucocytes (/field) >25 >25 >25 >25 NA NA NA Quantification of A. x culture (CFU/mL) 105 105 105 106 103 103 103 Other bacteria (CFU/mL) None None None None 108 M. c None 106 S. a A. x: Achromobacter xylosoxidans; M. c: Moraxella catarrhalis; NA: non available; PMN: Polymorphonuclear; RBC: red blood cells; S. a: Staphylococcus aureus. : BAL number in bold indicates BALs for which qPCR had been carried out. viruses-13-00060-t002_Table 2Table 2 Eight Achromobacter xylosoxidans isolates for which whole genome sequencing was carried out. These were isolated from bronchoalveolar lavages in a 12-year-old boy after double lung transplantation. The patient received a first round of phage administration on 8 September 2017. A second round of phage treatment was performed on 23 January 2018. Complete Name Isolate Strain Source of Sampling Date of Sampling Whole Genome Sequence is1S-Str1/BAL#3 is1S Str1 BAL#3 25 July 2017 ERS5044236-UGAX1 is2S-Str2/BAL#5 is2S Str2 BAL#5 15 September 2017 ERS5044237-UGAX2 is3R-Str1/BAL#5 is3R Str1 BAL#5 15 September 2017 ERS5044238-UGAX3 is5S-Str3/BAL#6 is5S Str3 BAL#6 12 October 2017 ERS5044239-UGAX5 is6S-Str3/BAL#6 is6S Str3 BAL#6 12 October 2017 ERS5044240-UGAX6 is7R-Str4/BAL#9 is7R Str4 BAL#9 8 February 2018 ERS5044241-UGAX7 is8R-Str4/BAL#9 is8R Str4 BAL#9 8 February 2018 ERS5044242-UGAX8 is9S-Str3/BAL#9 is9S Str3 BAL#9 8 February 2018 ERS5044243-UGAX9 * Submitted at ENA, 1 September 2020. Study ID: PRJEB39103. BAL: Bronchoalveolar lavage, S: Phage susceptible, R: phage resistant. viruses-13-00060-t003_Table 3Table 3 Results of bacterial culture (grey-shading) and qPCR of three broncho-alveolar lavages for four different Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. Bronchoalveolar Lavage BAL#5 BAL#6 BAL#9 15 September 2017 12 October 2017 8 February 2018 qPCR for Strain Isolates Str1 is1S, is3R N NS P (34.7) Str2 is2S P (36.6) N N Str3 is5S, is6S, is9S P (32.7) P (28.9) P (27.7) Str4 is7R, is8R NS NS P (35.2) Grey-shaded box = isolate cultured from this BAL; N: negative (Cq-value > 40); NS: Nonspecific amplification (melting peak different from that of specific amplification); P: positive qPCR (Cq-value).	Oral	DrugAdministrationRoute	CC BY	33466377	19,957,163	2021-01-05
What was the dosage of drug 'MYCOPHENOLATE MOFETIL'?	A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient. Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains. 1. Case Presentation We report the case of a 12-year-old boy (35.5 kg), who received double lung transplantation in 25 March 2017, (at the age of 12) because of cystic fibrosis. Before transplantation, he was colonized with Aspergillus fumigatus, Achromobacter xylosoxidans (first colonization identified in 2013), and Pseudomonas aeruginosa. After transplantation, he experienced acute kidney injury requiring temporary haemodialysis, pulmonary embolism, and persisting airway colonization with A. fumigatus. He was discharged on 29 April 2017, with satisfactory respiratory status; he initially required no home oxygen therapy. His immunosuppressive regimen included prednisolone (25 mg/day), tacrolimus (1.5 mg bis in die (b.i.d.) with a target trough concentration of 6–8 ng/mL), mycophenolate mofetil (1 g b.i.d.) and his antimicrobial prophylaxis was cotrimoxazole (400/80 mg, three times a week), posaconazole (150 mg, oral suspension, ter in die (t.i.d.)), valacyclovir (500 mg b.i.d.), and intravenous immunoglobulins (15 g each three weeks). Between May and June 2017, he experienced progressive shortness of breath, cough and increased sputum production; he subsequently required the addition of 1–1.5 L/min oxygen therapy. Because of a stenosis of the surgical anastomosis of the main left bronchus, a dilatation during rigid bronchoscopy was performed on 19 May and a Montgomery stent was inserted on 2 June. Later on, a stenosis of the surgical anastomosis on the right bronchus intermedius required a dilatation during rigid bronchoscopy followed by the insertion of an Oki stent on 29 June. Several bronchoalveolar lavages (BAL), performed between May and June 2017, revealed inflammation (between 800 and >1000 leukocytes/mm3 with 88–90% of polymorphonuclear cells) and repeatedly grew pandrug-resistant A. xylosoxidans (Table 1 and Supplementary Figure S1) [1]. Lung biopsy performed on 19 June was consistent with lung infection: diffuse acute bronchiolitis with extension to peri-bronchiolar alveoli. Microbiological samples obtained from lung biopsies were negative (bacterial, mycological, and mycobacteriological cultures). The first antibiotic treatment consisted of intravenous tigecycline 50 mg b.i.d. from 29 June to 31 July. As BAL remained positive for A. xylosoxidans and the patient still required oxygen therapy; antibiotic treatment was subsequently switched to imipenem (1200 mg, t.i.d., ~100 mg/Kg/d) on 31 July 2017. Subsequent respiratory samples still grew A. xylosoxidans and the respiratory status did not improve (requiring oxygen at home, 1 L/min). Furthermore, despite antibiotic treatment and correct drainage of the right superior lobe, lung consolidation and micronodules remained. Considering the failure of antibiotic treatment, phage therapy was proposed to the patient and his family, after multidisciplinary discussion. A first cocktail (APC 1.1) containing three lytic phages (JWDelta, JWT and 2-1) active against A. xylosoxidans isolate is1S (designated such as later), selected from the DSMZ collection (Braunschweig, Germany) [2,3], was prepared at the Laboratory for Bacteriology Research (Ghent University, Belgium) and the Laboratory for Molecular and Cellular Technology (Queen Astrid Military Hospital, Brussels, Belgium) using the above-mentioned bacterial isolate, as previously described [4]. The production was completed on 23 August with a final phage titer of 4 × 1010 plaque forming units (pfu)/mL defined by double-agar overlay method [4], an endotoxin level of 4000 EU/mL (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, GenScript, Piscataway Township, NJ, USA) and a pH of 7.3 (in sterile PBS). Regulatory permission for phage importation was obtained on 1 September 2017, from the Agence Nationale de Sécurité du Médicament (ANSM). A first round of phage administration was performed, consisting of 3 nebulizations/day of 5 mL of the filter-sterilized (Sartorius 0.2 µm) phage solution, tenfold diluted in sterile saline on 8 and 9 September, using a vibrating mesh nebulizer (eFLOW rapid, PARI, PARI GmbH, Germany) with a mouth piece. Immediate tolerance was perfect, but no clinical improvement was noted and culture of subsequent respiratory samples remained positive for A. xylosoxidans (104 CFU/mL in the BAL of 15 September). A second cocktail (APC 2.1) was produced, in which phage JWalpha was added to the three lytic phages of cocktail APC 1.1, to improve the therapeutic efficacy. The production was completed on 22 December with a final phage titer of 5 × 109 pfu/mL, an endotoxin level of 1760 EU/mL and a pH of 6.95 (in sterile PBS). Regulatory permission was obtained for phage importation on 16 January 2018 (ANSM). On 23 January, during therapeutic bronchoscopy under general anesthesia, 30 mL of APC2.1, tenfold diluted (in sterile saline) and filter-sterilized, was instilled in each pulmonary lobe through the fibroscope. Immediate tolerance was perfect and the patient was discharged on 24 January with continued phage nebulization at home: three times a day 5 mL of preparation A using a vibrating mesh nebulizer (eFLOW rapid, PARI) with a mouth piece for 14 days until 6 February. Clinical tolerance was perfect again, but the initial clinical status remained unchanged. BAL performed on 8 February 2018, two days after stopping the phage treatment, still grew 105 cfu/mL of A. xylosoxidans. Subsequently, the patient’s respiratory condition slowly improved and oxygen therapy was stopped on 15 February 2018. No clinical worsening was observed after imipenem interruption on 16 February 2018. Sputum culture remained positive for A. xylosoxidans but with a low bacterial density (103 CFU/mL in March and May 2018, and June 2019) and no A. xylosoxidans could be isolated from BALs#10 and #11, sampled on August 2019, and April 2020, respectively (Table 1). The last pulmonary function tests, performed on 17 October 2019, showed best results since lung transplantation, with an FEV1 of 79% and a forced vital capacity of 85%. 2. Microbiological Analysis of Eight A. xylosoxidans Isolates As we observed discrepant outcome in our patient (clinical improvement on the one hand but persisting low-grade airway colonization on the other hand), we decided to study the eight available A. xylosoxidans isolates (is1 to is9) from the patient’s colonization obtained by random picking from the culture plates from 4 distinct BALs (Table 2). These eight isolates, taken before, during and after the phage therapy period (Table 2), could not be differentiated from each other by means of MALDI-TOF [5] or McRAPD typing [6] and all belonged to the epidemic clone, already described previously among Belgian CF patients [5]. Whole genome sequencing (WGS) and de novo genome assembly for these eight isolates was carried out (Supplementary Methods and Table S1). The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Total genome length for all eight isolates was 6.44–6.50 Mbp, with a GC content of 67.5%. All eight isolates showed most similarity with reference genome GCF_008432465 (AX1), which corresponds to the type strain of A. xylosoxidans subsp. xylosoxidans (ATCC 27061T, CCUG 12689T, DSMZ 10346T, LMG 1863T, and NCTC 10807T), i.e., between 88.38 and 89.60% on the basis of mapped sequence reads, and between 98.71% and 98.76% on the basis of average nucleotide identity (ANI). Indeed, the overall similarity between the genome sequences of the eight isolates was high, i.e., 99.60%, as assessed by digital DNA hybridization, and more than 99.97% according to ANI-analysis. This was confirmed by variant calling where we observed that the isolates differed from each other only for a total of 367 SNPs, whereas a total of 59,452 SNPs were observed between the isolates and the best matching reference genome (AX1) (Figure 1). The susceptibility phenotype of each of the eight isolates was assessed by spot-test against the phage cocktail APC 2.1 and expressed as “S” when susceptible or “R” when resistant (e.g., “is2S” means that isolate number 2 was susceptible to the phage cocktail) (Table 2 and Supplementary Methods). Among these eight isolates, analysis based on variant calling furthermore revealed four closely related but clearly delineated strains, i.e., clones that differ genetically from other clones, with different phage susceptibility phenotype (Table 2 and Figure 1): Strain 1 (Str1), contained isolates 1 and 3 (is1S and is3R), found in BAL#3 (is1S-Str1/BAL#3) and BAL#5 (is3R-Str1/BAL#5), respectively. Strain 2 (Str2) contained isolate 2 (is2S) from BAL#5 (is2S-Str2/BAL#5). Strain 3 (Str3) contained isolates 5, 6, and 9 (is5S, is6S and is9S), which were found in BAL#6 (is5S-Str3/BAL#6, is6S-Str3/BAL#6) and BAL#9 (is9S-Str3/BAL#9). Strain 4 contained isolates 7 and 8 (is7R and is8R), found in BAL#9 (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) (Table 2). Isolate is1S-Str1/BAL#3 was obtained on 25 July 2017, i.e., 45 days before the first phage therapy session (8 September 2017) and was 100% identical to is3R-Str1/BAL#5 isolated seven days (15 September 2017) after the first phage therapy session (Figure 1). Since both isolates is1S-Str1/BAL#3 and is3R-Str1/BAL#5 are 100% identical, this means that the phage resistance mechanism of isolate is3R-Str1/BAL#5 is not genetically supported (see discussion). Isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6, both phage susceptible, were obtained one month later (12 October 2017) and belonged to still another strain (Strain 3). Finally, on 8 February 2018, i.e., 16 days after the second phage therapy session (23 January 2018), an additional three isolates were obtained from BAL#9. Of these, isolate is9S-Str3/BAL#9 belonged to the same Strain 3 as isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6 and was phage susceptible as well, but isolates 7R and 8R belonged to still another strain (Strain 4) and were both resistant to the phage cocktail. Interestingly, the phage resistant Strain 4 isolates (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) contained mutations compared to the other six isolates that could explain the phage resistance observed for these two isolates (Figure 1): when Strain 1 was used as a reference, a C to A transition at position 1803 of the colicin I receptor gene caused a Tyr601->Stop transition. This receptor has been recognized as a phage receptor [7]. We further studied the course of infection by extracting DNA from the three available A. xylosoxidans-positive BAL samples (BAL#5, #6 and #9), and by quantifying DNA of the different strains by means of strain-specific qPCRs (Table 3 and Supplementary Methods) that had been developed on the basis of the obtained WGS data. qPCRs for Strain 2 and Strain 4 were only positive for the BALs from which these isolates had been cultured, respectively, BAL#5 and BAL#9. qPCR for Strain 1 was only positive for BAL#9, but not for BAL#5 from which isolate 3R had been cultured. qPCR for Strain 3 was positive for all three BALs, although no isolate belonging to Strain 3 was cultured from BAL#5. 3. Discussion and Conclusions We report the first case of decolonization of pandrug-resistant Achromobacter xylosoxidans after a long-term follow-up in a lung-transplanted patient receiving phage therapy. The first actual demonstration and usage of bacteriophages came almost simultaneously from two microbiologists in 1915 (Frederick Twort) and 1917 (Félix d’Hérelle) [8]. After extensive clinical use during the first half of the 20th century, their use has been progressively reduced, mainly due to the discovery, rapid spread, and ease of use of antibiotics for the treatment of infectious diseases [9]. However, bacteriophages gained a new surge of attention, due to the emergence and spread of pandrug-resistant bacteria [8]. Despite decades of clinical experience with bacteriophages in Eastern Europe, very few data are available regarding their clinical efficacy; besides, case-series and few randomized controlled trials (RCT) [10,11,12]. For instance, a RCT assessed the clinical outcome of patients receiving local application of a phage cocktail for the treatment of burn wound infection. This RCT is frequently cited to have shown no clinical benefits, as compared to standard of care. However, the phage titer that was applied (100 pfu/mL) [10], was so extremely low that it precludes to draw any conclusion from this study regarding the usefulness of phage therapy. The limited number of randomized clinical trials has been recently reviewed [13]. Phage therapy has also been used locally, e.g., for the treatment of bone and joint infections [14,15] or intravenously for the treatment of bloodstream infections [16,17]. Considering the high frequency of colonization and infection caused by antibiotic-resistant bacteria among cystic fibrosis and lung-transplanted patients, such as in our patient, the use of bacteriophages appears to be an appealing option in this population [18,19,20]. In the specific case of A. xylosoxidans, thus far a single case-report described the favorable outcome after the use of bacteriophages in a cystic fibrosis patient [21]. Our case-report highlights the many difficulties that need to be taken into account before considering the use of bacteriophages in a clinical setting. These include amongst others an important delay from clinical decision to regulatory agency authorization. Most problematic is the difficult interpretation of the clinical outcome after bacteriophage therapy, considering the long-term persisting airway colonization and the possible evolution of different A. xylosoxidans strains. The second therapeutic attempt relied on a massive lung instillation (injection of the bacteriophage solution in the patient’s lung during fibroscopy) followed by a two-week course of phage nebulization. We used a vibrating mesh nebulizer for phage delivery as this type of nebulizer has been shown to result in a lower phage titer reduction, as compared to jet nebulizers [22]. BAL#9, performed two weeks after the second round of phage therapy (8 February 2018) still grew 105 CFU/mL of A. xylosoxidans, represented by Strains 3 and 4 (according to culture) and by at least Strains 1, 3 and 4 (according to qPCR) (Table 1, Table 2 and Table 3). Sputum cultures remained positive for 18 months, but with only 103 CFU A. xylosoxidans/mL, suggesting a possible upper airway colonization, especially since no A. xylosoxidans could be isolated from BALs sampled in August 2019, and April 2020. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. To more precisely understand the discrepant outcome of our patient, another option would have been to search for active phages from the BAL samples throughout the study, but this was not performed. Despite the observed differences of a total of 367 SNPs between the four strains of eight A. xylosoxidans isolates from the patient, their overall similarity suggests that they originate from a single strain that colonized the patient (who was colonized with A. xylosoxidans years before the start of the phage therapy). Therefore, it is possible that some variants were present already before lung transplantation and before the start of phage therapy. Although it is not possible to exclude the simultaneous presence of these different strains on the basis of our culture results, because only one or two colonies were picked from each BAL culture, analysis by means of four strain-specific qPCR assays of the three BAL samples that were available for study, indicated that Strains 2 and 4 were probably prevalent at a particular moment of the course of infection, whereas Strains 1 and 3 could be identified during the full course of the infection. Phage resistant Strain 4 may have been selected during phage therapy. Phage training has been proposed to overcome phage resistance and increase infectivity against a given bacterial population [23]. However, training phages may take weeks, and therefore might be restricted to the treatment of chronic infections. Regarding more acute infections, the establishment of phage collections (such as the DSMZ, which provided bacteriophages for our patient) is of critical importance to provide phages active against a broad spectrum of bacterial isolates. An interesting point is the observation of two isolates from the same strain (is1S-Str1/BAL#3 and is3R-Str1/BAL#5) that differ regarding their phage susceptibility phenotype despite the absence of genetic support (identical genome). As observed with bacteria where emerging data point out that a resistance phenotype against a given antimicrobial may not be supported by a genotypic substratum [24,25], phage resistance without any change in genotype has also been documented [26]. These genotype/phenotype discrepancies are hypothesized to be possible through epigenetic regulation mechanisms, of which post-transcriptional regulation, which may influence phage receptor expression on the cell surface or phage receptor masking (through capsule synthesis). Further studies are required to better understand the dynamics of in vivo selection of phage-resistant bacteria. Because the patient’s medical condition improved anyhow, a few weeks after the second round of phage instillation, it is possible that phage therapy selected the phage-resistant A. xylosoxidans Strain 4, but that the mutation that caused phage resistance at the same time decreased the virulence of the strain as already described with other bacteria and phages [27,28,29]. Indeed, we observed several mutations which could explain the phage resistance of Strain 4, of which one was especially interesting, since it caused the gain of a stop codon in the colicin I receptor, which has been recognized as a phage receptor in other species [7]. Because in E. coli, the colicin I receptor plays a crucial role in iron transport across the outer membrane [7], loss of this function due to the observed mutation may have caused reduced fitness of the A. xylosoxidans mutant Strain 4. However, from a clinical point-of-view, one cannot exclude a spontaneous improvement, independently of phage therapy and despite months of fruitless antibiotic treatment, highlighting the dire need of randomized clinical trials in order to more convincingly assess the clinical efficacy of bacteriophages. In conclusion, we describe the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. Despite initial persisting airway colonization, the final clinical and microbiological outcome was favorable. Whole genome sequencing of eight A. xylosoxidans isolates and strain-specific qPCR experiments performed on BALs sampled before and after phage therapy allowed to more precisely study the dynamics of lung colonization by different isolates, susceptible or resistant to the applied phage cocktails. Acknowledgments The authors would like to thank L. Lécuyer and V. Boussaud (Unité de Transplantation, HEGP), B. Sabatier, V. Savoldelli, T. Caruba and P. Prognon (Pharmacie HEGP), P. Odou, C. Nassar, C. Berneron and M. Roche (Pharmacie Lille), and E. Deberranger (Hématologie Pédiatrique Lille), for their help in the management of the patient. Microbiological analysis was performed with a funding by the Association Robert Debré pour la Recherche Médicale. The authors would also like to thank Caroline Semaille and Nathalie Morgensztejn from the Agence Nationale de Sécurité du Médicament (ANSM) for their regulatory support. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Materials The following are available online at https://www.mdpi.com/1999-4915/13/1/60/s1, Figure S1: Antibiotic susceptibility testing (AST) of a pandrug-resistant Achromobacter xylosoxidans isolated during a bronchoalveolar lavage of a 12-year-old boy after double lung transplantation, Table S1: Variant calling for the eight Achromobacter xylosoxidans whole genome sequences, see excel file attached, named: “Supplementary Table S1. Variant calling.xls”. Click here for additional data file. Author Contributions D.L. (David Lebeaux) and M.V. conceptualized the study, validated the data, carried out formal analysis, prepared the initial draft, took care of project administration and supervised the study. Data were visualized by H.D. and Y.G. Software analysis was carried out by H.D., S.V., Y.G. D.L. (David Lebeaux), M.M., R.G., I.P., F.C., N.K., J.W., C.R., E.C., D.L. (Damien Lanoy), L.V.S., H.D., J.-P.P., N.D., S.V., Y.G., F.V.N., and M.V. contributed to basic methodology. Data curation was done by D.L. (David Lebeaux), M.M., C.T., S.V., Y.G., and M.V. Funding was acquired by D.L. (David Lebeaux), J.-L.M., F.V.N., and M.V. All authors took part in the investigation and in final reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement The study did not require ethical approval. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Phylogenetic tree built with Whole genome sequences of eight Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. The distance between samples reflects the number of different Single Nucleotide Polymorphisms (SNPs). Each A. xylosoxidans is named as follows: isolate number (is1 to is8) S (or R to depict susceptibility or resistance to phage cocktail)-Strain number/Number of BAL where it has been identified. viruses-13-00060-t001_Table 1Table 1 Results of bronchoalveolar lavages (BALs) and sputum analysis from May 2017, to April 2020, in 12-year-old lung transplanted patient who received two rounds of phage therapy for pandrug-resistant Achromobacter xylosoxidans colonization and infection. Date 19 May 2017 23 May 2017 6 June 2017 12 June 2017 25 July 2017 5 September 2017 8 September 2017 15 September 2017 12 October 2017 27 October 2017 23 January 2018 8 February 2018 5 March 2018 18 May 2018 19 February 2019 19 June 2019 27 August 2019 30 April 2020 Phage therapy ROUND 1 ROUND 2 Tigecycline from 29 June to 31 July Imipenem From 31 July 2017, to 16 February 2018 Number of days, relative to the first round of phage therapy −112 −108 −94 −88 −45 −3 +7 +37 +49 +137 +153 +178 +252 +529 +649 +718 +963 BAL BAL Number * #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 Leukocytes (/mm3) >1000 800 >1000 500 >1000 60 >1000 810 >1000 NA NA NA PMN cells (%) 90 88 90 91 43 30 80 69 82 NA NA NA Quantification of A. x culture (CFU/mL) 104 103 104 103 104 103 104 106 105 104 0 0 Other bacteria (CFU/mL) None None None None None None None None None None 104 S. a 106 S. a Sputum Leucocytes (/field) >25 >25 >25 >25 NA NA NA Quantification of A. x culture (CFU/mL) 105 105 105 106 103 103 103 Other bacteria (CFU/mL) None None None None 108 M. c None 106 S. a A. x: Achromobacter xylosoxidans; M. c: Moraxella catarrhalis; NA: non available; PMN: Polymorphonuclear; RBC: red blood cells; S. a: Staphylococcus aureus. : BAL number in bold indicates BALs for which qPCR had been carried out. viruses-13-00060-t002_Table 2Table 2 Eight Achromobacter xylosoxidans isolates for which whole genome sequencing was carried out. These were isolated from bronchoalveolar lavages in a 12-year-old boy after double lung transplantation. The patient received a first round of phage administration on 8 September 2017. A second round of phage treatment was performed on 23 January 2018. Complete Name Isolate Strain Source of Sampling Date of Sampling Whole Genome Sequence is1S-Str1/BAL#3 is1S Str1 BAL#3 25 July 2017 ERS5044236-UGAX1 is2S-Str2/BAL#5 is2S Str2 BAL#5 15 September 2017 ERS5044237-UGAX2 is3R-Str1/BAL#5 is3R Str1 BAL#5 15 September 2017 ERS5044238-UGAX3 is5S-Str3/BAL#6 is5S Str3 BAL#6 12 October 2017 ERS5044239-UGAX5 is6S-Str3/BAL#6 is6S Str3 BAL#6 12 October 2017 ERS5044240-UGAX6 is7R-Str4/BAL#9 is7R Str4 BAL#9 8 February 2018 ERS5044241-UGAX7 is8R-Str4/BAL#9 is8R Str4 BAL#9 8 February 2018 ERS5044242-UGAX8 is9S-Str3/BAL#9 is9S Str3 BAL#9 8 February 2018 ERS5044243-UGAX9 * Submitted at ENA, 1 September 2020. Study ID: PRJEB39103. BAL: Bronchoalveolar lavage, S: Phage susceptible, R: phage resistant. viruses-13-00060-t003_Table 3Table 3 Results of bacterial culture (grey-shading) and qPCR of three broncho-alveolar lavages for four different Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. Bronchoalveolar Lavage BAL#5 BAL#6 BAL#9 15 September 2017 12 October 2017 8 February 2018 qPCR for Strain Isolates Str1 is1S, is3R N NS P (34.7) Str2 is2S P (36.6) N N Str3 is5S, is6S, is9S P (32.7) P (28.9) P (27.7) Str4 is7R, is8R NS NS P (35.2) Grey-shaded box = isolate cultured from this BAL; N: negative (Cq-value > 40); NS: Nonspecific amplification (melting peak different from that of specific amplification); P: positive qPCR (Cq-value).	1 g (grams).	DrugDosage	CC BY	33466377	19,957,163	2021-01-05
What was the dosage of drug 'POSACONAZOLE'?	A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient. Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains. 1. Case Presentation We report the case of a 12-year-old boy (35.5 kg), who received double lung transplantation in 25 March 2017, (at the age of 12) because of cystic fibrosis. Before transplantation, he was colonized with Aspergillus fumigatus, Achromobacter xylosoxidans (first colonization identified in 2013), and Pseudomonas aeruginosa. After transplantation, he experienced acute kidney injury requiring temporary haemodialysis, pulmonary embolism, and persisting airway colonization with A. fumigatus. He was discharged on 29 April 2017, with satisfactory respiratory status; he initially required no home oxygen therapy. His immunosuppressive regimen included prednisolone (25 mg/day), tacrolimus (1.5 mg bis in die (b.i.d.) with a target trough concentration of 6–8 ng/mL), mycophenolate mofetil (1 g b.i.d.) and his antimicrobial prophylaxis was cotrimoxazole (400/80 mg, three times a week), posaconazole (150 mg, oral suspension, ter in die (t.i.d.)), valacyclovir (500 mg b.i.d.), and intravenous immunoglobulins (15 g each three weeks). Between May and June 2017, he experienced progressive shortness of breath, cough and increased sputum production; he subsequently required the addition of 1–1.5 L/min oxygen therapy. Because of a stenosis of the surgical anastomosis of the main left bronchus, a dilatation during rigid bronchoscopy was performed on 19 May and a Montgomery stent was inserted on 2 June. Later on, a stenosis of the surgical anastomosis on the right bronchus intermedius required a dilatation during rigid bronchoscopy followed by the insertion of an Oki stent on 29 June. Several bronchoalveolar lavages (BAL), performed between May and June 2017, revealed inflammation (between 800 and >1000 leukocytes/mm3 with 88–90% of polymorphonuclear cells) and repeatedly grew pandrug-resistant A. xylosoxidans (Table 1 and Supplementary Figure S1) [1]. Lung biopsy performed on 19 June was consistent with lung infection: diffuse acute bronchiolitis with extension to peri-bronchiolar alveoli. Microbiological samples obtained from lung biopsies were negative (bacterial, mycological, and mycobacteriological cultures). The first antibiotic treatment consisted of intravenous tigecycline 50 mg b.i.d. from 29 June to 31 July. As BAL remained positive for A. xylosoxidans and the patient still required oxygen therapy; antibiotic treatment was subsequently switched to imipenem (1200 mg, t.i.d., ~100 mg/Kg/d) on 31 July 2017. Subsequent respiratory samples still grew A. xylosoxidans and the respiratory status did not improve (requiring oxygen at home, 1 L/min). Furthermore, despite antibiotic treatment and correct drainage of the right superior lobe, lung consolidation and micronodules remained. Considering the failure of antibiotic treatment, phage therapy was proposed to the patient and his family, after multidisciplinary discussion. A first cocktail (APC 1.1) containing three lytic phages (JWDelta, JWT and 2-1) active against A. xylosoxidans isolate is1S (designated such as later), selected from the DSMZ collection (Braunschweig, Germany) [2,3], was prepared at the Laboratory for Bacteriology Research (Ghent University, Belgium) and the Laboratory for Molecular and Cellular Technology (Queen Astrid Military Hospital, Brussels, Belgium) using the above-mentioned bacterial isolate, as previously described [4]. The production was completed on 23 August with a final phage titer of 4 × 1010 plaque forming units (pfu)/mL defined by double-agar overlay method [4], an endotoxin level of 4000 EU/mL (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, GenScript, Piscataway Township, NJ, USA) and a pH of 7.3 (in sterile PBS). Regulatory permission for phage importation was obtained on 1 September 2017, from the Agence Nationale de Sécurité du Médicament (ANSM). A first round of phage administration was performed, consisting of 3 nebulizations/day of 5 mL of the filter-sterilized (Sartorius 0.2 µm) phage solution, tenfold diluted in sterile saline on 8 and 9 September, using a vibrating mesh nebulizer (eFLOW rapid, PARI, PARI GmbH, Germany) with a mouth piece. Immediate tolerance was perfect, but no clinical improvement was noted and culture of subsequent respiratory samples remained positive for A. xylosoxidans (104 CFU/mL in the BAL of 15 September). A second cocktail (APC 2.1) was produced, in which phage JWalpha was added to the three lytic phages of cocktail APC 1.1, to improve the therapeutic efficacy. The production was completed on 22 December with a final phage titer of 5 × 109 pfu/mL, an endotoxin level of 1760 EU/mL and a pH of 6.95 (in sterile PBS). Regulatory permission was obtained for phage importation on 16 January 2018 (ANSM). On 23 January, during therapeutic bronchoscopy under general anesthesia, 30 mL of APC2.1, tenfold diluted (in sterile saline) and filter-sterilized, was instilled in each pulmonary lobe through the fibroscope. Immediate tolerance was perfect and the patient was discharged on 24 January with continued phage nebulization at home: three times a day 5 mL of preparation A using a vibrating mesh nebulizer (eFLOW rapid, PARI) with a mouth piece for 14 days until 6 February. Clinical tolerance was perfect again, but the initial clinical status remained unchanged. BAL performed on 8 February 2018, two days after stopping the phage treatment, still grew 105 cfu/mL of A. xylosoxidans. Subsequently, the patient’s respiratory condition slowly improved and oxygen therapy was stopped on 15 February 2018. No clinical worsening was observed after imipenem interruption on 16 February 2018. Sputum culture remained positive for A. xylosoxidans but with a low bacterial density (103 CFU/mL in March and May 2018, and June 2019) and no A. xylosoxidans could be isolated from BALs#10 and #11, sampled on August 2019, and April 2020, respectively (Table 1). The last pulmonary function tests, performed on 17 October 2019, showed best results since lung transplantation, with an FEV1 of 79% and a forced vital capacity of 85%. 2. Microbiological Analysis of Eight A. xylosoxidans Isolates As we observed discrepant outcome in our patient (clinical improvement on the one hand but persisting low-grade airway colonization on the other hand), we decided to study the eight available A. xylosoxidans isolates (is1 to is9) from the patient’s colonization obtained by random picking from the culture plates from 4 distinct BALs (Table 2). These eight isolates, taken before, during and after the phage therapy period (Table 2), could not be differentiated from each other by means of MALDI-TOF [5] or McRAPD typing [6] and all belonged to the epidemic clone, already described previously among Belgian CF patients [5]. Whole genome sequencing (WGS) and de novo genome assembly for these eight isolates was carried out (Supplementary Methods and Table S1). The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Total genome length for all eight isolates was 6.44–6.50 Mbp, with a GC content of 67.5%. All eight isolates showed most similarity with reference genome GCF_008432465 (AX1), which corresponds to the type strain of A. xylosoxidans subsp. xylosoxidans (ATCC 27061T, CCUG 12689T, DSMZ 10346T, LMG 1863T, and NCTC 10807T), i.e., between 88.38 and 89.60% on the basis of mapped sequence reads, and between 98.71% and 98.76% on the basis of average nucleotide identity (ANI). Indeed, the overall similarity between the genome sequences of the eight isolates was high, i.e., 99.60%, as assessed by digital DNA hybridization, and more than 99.97% according to ANI-analysis. This was confirmed by variant calling where we observed that the isolates differed from each other only for a total of 367 SNPs, whereas a total of 59,452 SNPs were observed between the isolates and the best matching reference genome (AX1) (Figure 1). The susceptibility phenotype of each of the eight isolates was assessed by spot-test against the phage cocktail APC 2.1 and expressed as “S” when susceptible or “R” when resistant (e.g., “is2S” means that isolate number 2 was susceptible to the phage cocktail) (Table 2 and Supplementary Methods). Among these eight isolates, analysis based on variant calling furthermore revealed four closely related but clearly delineated strains, i.e., clones that differ genetically from other clones, with different phage susceptibility phenotype (Table 2 and Figure 1): Strain 1 (Str1), contained isolates 1 and 3 (is1S and is3R), found in BAL#3 (is1S-Str1/BAL#3) and BAL#5 (is3R-Str1/BAL#5), respectively. Strain 2 (Str2) contained isolate 2 (is2S) from BAL#5 (is2S-Str2/BAL#5). Strain 3 (Str3) contained isolates 5, 6, and 9 (is5S, is6S and is9S), which were found in BAL#6 (is5S-Str3/BAL#6, is6S-Str3/BAL#6) and BAL#9 (is9S-Str3/BAL#9). Strain 4 contained isolates 7 and 8 (is7R and is8R), found in BAL#9 (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) (Table 2). Isolate is1S-Str1/BAL#3 was obtained on 25 July 2017, i.e., 45 days before the first phage therapy session (8 September 2017) and was 100% identical to is3R-Str1/BAL#5 isolated seven days (15 September 2017) after the first phage therapy session (Figure 1). Since both isolates is1S-Str1/BAL#3 and is3R-Str1/BAL#5 are 100% identical, this means that the phage resistance mechanism of isolate is3R-Str1/BAL#5 is not genetically supported (see discussion). Isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6, both phage susceptible, were obtained one month later (12 October 2017) and belonged to still another strain (Strain 3). Finally, on 8 February 2018, i.e., 16 days after the second phage therapy session (23 January 2018), an additional three isolates were obtained from BAL#9. Of these, isolate is9S-Str3/BAL#9 belonged to the same Strain 3 as isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6 and was phage susceptible as well, but isolates 7R and 8R belonged to still another strain (Strain 4) and were both resistant to the phage cocktail. Interestingly, the phage resistant Strain 4 isolates (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) contained mutations compared to the other six isolates that could explain the phage resistance observed for these two isolates (Figure 1): when Strain 1 was used as a reference, a C to A transition at position 1803 of the colicin I receptor gene caused a Tyr601->Stop transition. This receptor has been recognized as a phage receptor [7]. We further studied the course of infection by extracting DNA from the three available A. xylosoxidans-positive BAL samples (BAL#5, #6 and #9), and by quantifying DNA of the different strains by means of strain-specific qPCRs (Table 3 and Supplementary Methods) that had been developed on the basis of the obtained WGS data. qPCRs for Strain 2 and Strain 4 were only positive for the BALs from which these isolates had been cultured, respectively, BAL#5 and BAL#9. qPCR for Strain 1 was only positive for BAL#9, but not for BAL#5 from which isolate 3R had been cultured. qPCR for Strain 3 was positive for all three BALs, although no isolate belonging to Strain 3 was cultured from BAL#5. 3. Discussion and Conclusions We report the first case of decolonization of pandrug-resistant Achromobacter xylosoxidans after a long-term follow-up in a lung-transplanted patient receiving phage therapy. The first actual demonstration and usage of bacteriophages came almost simultaneously from two microbiologists in 1915 (Frederick Twort) and 1917 (Félix d’Hérelle) [8]. After extensive clinical use during the first half of the 20th century, their use has been progressively reduced, mainly due to the discovery, rapid spread, and ease of use of antibiotics for the treatment of infectious diseases [9]. However, bacteriophages gained a new surge of attention, due to the emergence and spread of pandrug-resistant bacteria [8]. Despite decades of clinical experience with bacteriophages in Eastern Europe, very few data are available regarding their clinical efficacy; besides, case-series and few randomized controlled trials (RCT) [10,11,12]. For instance, a RCT assessed the clinical outcome of patients receiving local application of a phage cocktail for the treatment of burn wound infection. This RCT is frequently cited to have shown no clinical benefits, as compared to standard of care. However, the phage titer that was applied (100 pfu/mL) [10], was so extremely low that it precludes to draw any conclusion from this study regarding the usefulness of phage therapy. The limited number of randomized clinical trials has been recently reviewed [13]. Phage therapy has also been used locally, e.g., for the treatment of bone and joint infections [14,15] or intravenously for the treatment of bloodstream infections [16,17]. Considering the high frequency of colonization and infection caused by antibiotic-resistant bacteria among cystic fibrosis and lung-transplanted patients, such as in our patient, the use of bacteriophages appears to be an appealing option in this population [18,19,20]. In the specific case of A. xylosoxidans, thus far a single case-report described the favorable outcome after the use of bacteriophages in a cystic fibrosis patient [21]. Our case-report highlights the many difficulties that need to be taken into account before considering the use of bacteriophages in a clinical setting. These include amongst others an important delay from clinical decision to regulatory agency authorization. Most problematic is the difficult interpretation of the clinical outcome after bacteriophage therapy, considering the long-term persisting airway colonization and the possible evolution of different A. xylosoxidans strains. The second therapeutic attempt relied on a massive lung instillation (injection of the bacteriophage solution in the patient’s lung during fibroscopy) followed by a two-week course of phage nebulization. We used a vibrating mesh nebulizer for phage delivery as this type of nebulizer has been shown to result in a lower phage titer reduction, as compared to jet nebulizers [22]. BAL#9, performed two weeks after the second round of phage therapy (8 February 2018) still grew 105 CFU/mL of A. xylosoxidans, represented by Strains 3 and 4 (according to culture) and by at least Strains 1, 3 and 4 (according to qPCR) (Table 1, Table 2 and Table 3). Sputum cultures remained positive for 18 months, but with only 103 CFU A. xylosoxidans/mL, suggesting a possible upper airway colonization, especially since no A. xylosoxidans could be isolated from BALs sampled in August 2019, and April 2020. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. To more precisely understand the discrepant outcome of our patient, another option would have been to search for active phages from the BAL samples throughout the study, but this was not performed. Despite the observed differences of a total of 367 SNPs between the four strains of eight A. xylosoxidans isolates from the patient, their overall similarity suggests that they originate from a single strain that colonized the patient (who was colonized with A. xylosoxidans years before the start of the phage therapy). Therefore, it is possible that some variants were present already before lung transplantation and before the start of phage therapy. Although it is not possible to exclude the simultaneous presence of these different strains on the basis of our culture results, because only one or two colonies were picked from each BAL culture, analysis by means of four strain-specific qPCR assays of the three BAL samples that were available for study, indicated that Strains 2 and 4 were probably prevalent at a particular moment of the course of infection, whereas Strains 1 and 3 could be identified during the full course of the infection. Phage resistant Strain 4 may have been selected during phage therapy. Phage training has been proposed to overcome phage resistance and increase infectivity against a given bacterial population [23]. However, training phages may take weeks, and therefore might be restricted to the treatment of chronic infections. Regarding more acute infections, the establishment of phage collections (such as the DSMZ, which provided bacteriophages for our patient) is of critical importance to provide phages active against a broad spectrum of bacterial isolates. An interesting point is the observation of two isolates from the same strain (is1S-Str1/BAL#3 and is3R-Str1/BAL#5) that differ regarding their phage susceptibility phenotype despite the absence of genetic support (identical genome). As observed with bacteria where emerging data point out that a resistance phenotype against a given antimicrobial may not be supported by a genotypic substratum [24,25], phage resistance without any change in genotype has also been documented [26]. These genotype/phenotype discrepancies are hypothesized to be possible through epigenetic regulation mechanisms, of which post-transcriptional regulation, which may influence phage receptor expression on the cell surface or phage receptor masking (through capsule synthesis). Further studies are required to better understand the dynamics of in vivo selection of phage-resistant bacteria. Because the patient’s medical condition improved anyhow, a few weeks after the second round of phage instillation, it is possible that phage therapy selected the phage-resistant A. xylosoxidans Strain 4, but that the mutation that caused phage resistance at the same time decreased the virulence of the strain as already described with other bacteria and phages [27,28,29]. Indeed, we observed several mutations which could explain the phage resistance of Strain 4, of which one was especially interesting, since it caused the gain of a stop codon in the colicin I receptor, which has been recognized as a phage receptor in other species [7]. Because in E. coli, the colicin I receptor plays a crucial role in iron transport across the outer membrane [7], loss of this function due to the observed mutation may have caused reduced fitness of the A. xylosoxidans mutant Strain 4. However, from a clinical point-of-view, one cannot exclude a spontaneous improvement, independently of phage therapy and despite months of fruitless antibiotic treatment, highlighting the dire need of randomized clinical trials in order to more convincingly assess the clinical efficacy of bacteriophages. In conclusion, we describe the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. Despite initial persisting airway colonization, the final clinical and microbiological outcome was favorable. Whole genome sequencing of eight A. xylosoxidans isolates and strain-specific qPCR experiments performed on BALs sampled before and after phage therapy allowed to more precisely study the dynamics of lung colonization by different isolates, susceptible or resistant to the applied phage cocktails. Acknowledgments The authors would like to thank L. Lécuyer and V. Boussaud (Unité de Transplantation, HEGP), B. Sabatier, V. Savoldelli, T. Caruba and P. Prognon (Pharmacie HEGP), P. Odou, C. Nassar, C. Berneron and M. Roche (Pharmacie Lille), and E. Deberranger (Hématologie Pédiatrique Lille), for their help in the management of the patient. Microbiological analysis was performed with a funding by the Association Robert Debré pour la Recherche Médicale. The authors would also like to thank Caroline Semaille and Nathalie Morgensztejn from the Agence Nationale de Sécurité du Médicament (ANSM) for their regulatory support. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Materials The following are available online at https://www.mdpi.com/1999-4915/13/1/60/s1, Figure S1: Antibiotic susceptibility testing (AST) of a pandrug-resistant Achromobacter xylosoxidans isolated during a bronchoalveolar lavage of a 12-year-old boy after double lung transplantation, Table S1: Variant calling for the eight Achromobacter xylosoxidans whole genome sequences, see excel file attached, named: “Supplementary Table S1. Variant calling.xls”. Click here for additional data file. Author Contributions D.L. (David Lebeaux) and M.V. conceptualized the study, validated the data, carried out formal analysis, prepared the initial draft, took care of project administration and supervised the study. Data were visualized by H.D. and Y.G. Software analysis was carried out by H.D., S.V., Y.G. D.L. (David Lebeaux), M.M., R.G., I.P., F.C., N.K., J.W., C.R., E.C., D.L. (Damien Lanoy), L.V.S., H.D., J.-P.P., N.D., S.V., Y.G., F.V.N., and M.V. contributed to basic methodology. Data curation was done by D.L. (David Lebeaux), M.M., C.T., S.V., Y.G., and M.V. Funding was acquired by D.L. (David Lebeaux), J.-L.M., F.V.N., and M.V. All authors took part in the investigation and in final reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement The study did not require ethical approval. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Phylogenetic tree built with Whole genome sequences of eight Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. The distance between samples reflects the number of different Single Nucleotide Polymorphisms (SNPs). Each A. xylosoxidans is named as follows: isolate number (is1 to is8) S (or R to depict susceptibility or resistance to phage cocktail)-Strain number/Number of BAL where it has been identified. viruses-13-00060-t001_Table 1Table 1 Results of bronchoalveolar lavages (BALs) and sputum analysis from May 2017, to April 2020, in 12-year-old lung transplanted patient who received two rounds of phage therapy for pandrug-resistant Achromobacter xylosoxidans colonization and infection. Date 19 May 2017 23 May 2017 6 June 2017 12 June 2017 25 July 2017 5 September 2017 8 September 2017 15 September 2017 12 October 2017 27 October 2017 23 January 2018 8 February 2018 5 March 2018 18 May 2018 19 February 2019 19 June 2019 27 August 2019 30 April 2020 Phage therapy ROUND 1 ROUND 2 Tigecycline from 29 June to 31 July Imipenem From 31 July 2017, to 16 February 2018 Number of days, relative to the first round of phage therapy −112 −108 −94 −88 −45 −3 +7 +37 +49 +137 +153 +178 +252 +529 +649 +718 +963 BAL BAL Number * #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 Leukocytes (/mm3) >1000 800 >1000 500 >1000 60 >1000 810 >1000 NA NA NA PMN cells (%) 90 88 90 91 43 30 80 69 82 NA NA NA Quantification of A. x culture (CFU/mL) 104 103 104 103 104 103 104 106 105 104 0 0 Other bacteria (CFU/mL) None None None None None None None None None None 104 S. a 106 S. a Sputum Leucocytes (/field) >25 >25 >25 >25 NA NA NA Quantification of A. x culture (CFU/mL) 105 105 105 106 103 103 103 Other bacteria (CFU/mL) None None None None 108 M. c None 106 S. a A. x: Achromobacter xylosoxidans; M. c: Moraxella catarrhalis; NA: non available; PMN: Polymorphonuclear; RBC: red blood cells; S. a: Staphylococcus aureus. : BAL number in bold indicates BALs for which qPCR had been carried out. viruses-13-00060-t002_Table 2Table 2 Eight Achromobacter xylosoxidans isolates for which whole genome sequencing was carried out. These were isolated from bronchoalveolar lavages in a 12-year-old boy after double lung transplantation. The patient received a first round of phage administration on 8 September 2017. A second round of phage treatment was performed on 23 January 2018. Complete Name Isolate Strain Source of Sampling Date of Sampling Whole Genome Sequence is1S-Str1/BAL#3 is1S Str1 BAL#3 25 July 2017 ERS5044236-UGAX1 is2S-Str2/BAL#5 is2S Str2 BAL#5 15 September 2017 ERS5044237-UGAX2 is3R-Str1/BAL#5 is3R Str1 BAL#5 15 September 2017 ERS5044238-UGAX3 is5S-Str3/BAL#6 is5S Str3 BAL#6 12 October 2017 ERS5044239-UGAX5 is6S-Str3/BAL#6 is6S Str3 BAL#6 12 October 2017 ERS5044240-UGAX6 is7R-Str4/BAL#9 is7R Str4 BAL#9 8 February 2018 ERS5044241-UGAX7 is8R-Str4/BAL#9 is8R Str4 BAL#9 8 February 2018 ERS5044242-UGAX8 is9S-Str3/BAL#9 is9S Str3 BAL#9 8 February 2018 ERS5044243-UGAX9 * Submitted at ENA, 1 September 2020. Study ID: PRJEB39103. BAL: Bronchoalveolar lavage, S: Phage susceptible, R: phage resistant. viruses-13-00060-t003_Table 3Table 3 Results of bacterial culture (grey-shading) and qPCR of three broncho-alveolar lavages for four different Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. Bronchoalveolar Lavage BAL#5 BAL#6 BAL#9 15 September 2017 12 October 2017 8 February 2018 qPCR for Strain Isolates Str1 is1S, is3R N NS P (34.7) Str2 is2S P (36.6) N N Str3 is5S, is6S, is9S P (32.7) P (28.9) P (27.7) Str4 is7R, is8R NS NS P (35.2) Grey-shaded box = isolate cultured from this BAL; N: negative (Cq-value > 40); NS: Nonspecific amplification (melting peak different from that of specific amplification); P: positive qPCR (Cq-value).	150 mg (milligrams).	DrugDosage	CC BY	33466377	19,957,163	2021-01-05
What was the dosage of drug 'PREDNISOLONE'?	A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient. Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains. 1. Case Presentation We report the case of a 12-year-old boy (35.5 kg), who received double lung transplantation in 25 March 2017, (at the age of 12) because of cystic fibrosis. Before transplantation, he was colonized with Aspergillus fumigatus, Achromobacter xylosoxidans (first colonization identified in 2013), and Pseudomonas aeruginosa. After transplantation, he experienced acute kidney injury requiring temporary haemodialysis, pulmonary embolism, and persisting airway colonization with A. fumigatus. He was discharged on 29 April 2017, with satisfactory respiratory status; he initially required no home oxygen therapy. His immunosuppressive regimen included prednisolone (25 mg/day), tacrolimus (1.5 mg bis in die (b.i.d.) with a target trough concentration of 6–8 ng/mL), mycophenolate mofetil (1 g b.i.d.) and his antimicrobial prophylaxis was cotrimoxazole (400/80 mg, three times a week), posaconazole (150 mg, oral suspension, ter in die (t.i.d.)), valacyclovir (500 mg b.i.d.), and intravenous immunoglobulins (15 g each three weeks). Between May and June 2017, he experienced progressive shortness of breath, cough and increased sputum production; he subsequently required the addition of 1–1.5 L/min oxygen therapy. Because of a stenosis of the surgical anastomosis of the main left bronchus, a dilatation during rigid bronchoscopy was performed on 19 May and a Montgomery stent was inserted on 2 June. Later on, a stenosis of the surgical anastomosis on the right bronchus intermedius required a dilatation during rigid bronchoscopy followed by the insertion of an Oki stent on 29 June. Several bronchoalveolar lavages (BAL), performed between May and June 2017, revealed inflammation (between 800 and >1000 leukocytes/mm3 with 88–90% of polymorphonuclear cells) and repeatedly grew pandrug-resistant A. xylosoxidans (Table 1 and Supplementary Figure S1) [1]. Lung biopsy performed on 19 June was consistent with lung infection: diffuse acute bronchiolitis with extension to peri-bronchiolar alveoli. Microbiological samples obtained from lung biopsies were negative (bacterial, mycological, and mycobacteriological cultures). The first antibiotic treatment consisted of intravenous tigecycline 50 mg b.i.d. from 29 June to 31 July. As BAL remained positive for A. xylosoxidans and the patient still required oxygen therapy; antibiotic treatment was subsequently switched to imipenem (1200 mg, t.i.d., ~100 mg/Kg/d) on 31 July 2017. Subsequent respiratory samples still grew A. xylosoxidans and the respiratory status did not improve (requiring oxygen at home, 1 L/min). Furthermore, despite antibiotic treatment and correct drainage of the right superior lobe, lung consolidation and micronodules remained. Considering the failure of antibiotic treatment, phage therapy was proposed to the patient and his family, after multidisciplinary discussion. A first cocktail (APC 1.1) containing three lytic phages (JWDelta, JWT and 2-1) active against A. xylosoxidans isolate is1S (designated such as later), selected from the DSMZ collection (Braunschweig, Germany) [2,3], was prepared at the Laboratory for Bacteriology Research (Ghent University, Belgium) and the Laboratory for Molecular and Cellular Technology (Queen Astrid Military Hospital, Brussels, Belgium) using the above-mentioned bacterial isolate, as previously described [4]. The production was completed on 23 August with a final phage titer of 4 × 1010 plaque forming units (pfu)/mL defined by double-agar overlay method [4], an endotoxin level of 4000 EU/mL (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, GenScript, Piscataway Township, NJ, USA) and a pH of 7.3 (in sterile PBS). Regulatory permission for phage importation was obtained on 1 September 2017, from the Agence Nationale de Sécurité du Médicament (ANSM). A first round of phage administration was performed, consisting of 3 nebulizations/day of 5 mL of the filter-sterilized (Sartorius 0.2 µm) phage solution, tenfold diluted in sterile saline on 8 and 9 September, using a vibrating mesh nebulizer (eFLOW rapid, PARI, PARI GmbH, Germany) with a mouth piece. Immediate tolerance was perfect, but no clinical improvement was noted and culture of subsequent respiratory samples remained positive for A. xylosoxidans (104 CFU/mL in the BAL of 15 September). A second cocktail (APC 2.1) was produced, in which phage JWalpha was added to the three lytic phages of cocktail APC 1.1, to improve the therapeutic efficacy. The production was completed on 22 December with a final phage titer of 5 × 109 pfu/mL, an endotoxin level of 1760 EU/mL and a pH of 6.95 (in sterile PBS). Regulatory permission was obtained for phage importation on 16 January 2018 (ANSM). On 23 January, during therapeutic bronchoscopy under general anesthesia, 30 mL of APC2.1, tenfold diluted (in sterile saline) and filter-sterilized, was instilled in each pulmonary lobe through the fibroscope. Immediate tolerance was perfect and the patient was discharged on 24 January with continued phage nebulization at home: three times a day 5 mL of preparation A using a vibrating mesh nebulizer (eFLOW rapid, PARI) with a mouth piece for 14 days until 6 February. Clinical tolerance was perfect again, but the initial clinical status remained unchanged. BAL performed on 8 February 2018, two days after stopping the phage treatment, still grew 105 cfu/mL of A. xylosoxidans. Subsequently, the patient’s respiratory condition slowly improved and oxygen therapy was stopped on 15 February 2018. No clinical worsening was observed after imipenem interruption on 16 February 2018. Sputum culture remained positive for A. xylosoxidans but with a low bacterial density (103 CFU/mL in March and May 2018, and June 2019) and no A. xylosoxidans could be isolated from BALs#10 and #11, sampled on August 2019, and April 2020, respectively (Table 1). The last pulmonary function tests, performed on 17 October 2019, showed best results since lung transplantation, with an FEV1 of 79% and a forced vital capacity of 85%. 2. Microbiological Analysis of Eight A. xylosoxidans Isolates As we observed discrepant outcome in our patient (clinical improvement on the one hand but persisting low-grade airway colonization on the other hand), we decided to study the eight available A. xylosoxidans isolates (is1 to is9) from the patient’s colonization obtained by random picking from the culture plates from 4 distinct BALs (Table 2). These eight isolates, taken before, during and after the phage therapy period (Table 2), could not be differentiated from each other by means of MALDI-TOF [5] or McRAPD typing [6] and all belonged to the epidemic clone, already described previously among Belgian CF patients [5]. Whole genome sequencing (WGS) and de novo genome assembly for these eight isolates was carried out (Supplementary Methods and Table S1). The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Total genome length for all eight isolates was 6.44–6.50 Mbp, with a GC content of 67.5%. All eight isolates showed most similarity with reference genome GCF_008432465 (AX1), which corresponds to the type strain of A. xylosoxidans subsp. xylosoxidans (ATCC 27061T, CCUG 12689T, DSMZ 10346T, LMG 1863T, and NCTC 10807T), i.e., between 88.38 and 89.60% on the basis of mapped sequence reads, and between 98.71% and 98.76% on the basis of average nucleotide identity (ANI). Indeed, the overall similarity between the genome sequences of the eight isolates was high, i.e., 99.60%, as assessed by digital DNA hybridization, and more than 99.97% according to ANI-analysis. This was confirmed by variant calling where we observed that the isolates differed from each other only for a total of 367 SNPs, whereas a total of 59,452 SNPs were observed between the isolates and the best matching reference genome (AX1) (Figure 1). The susceptibility phenotype of each of the eight isolates was assessed by spot-test against the phage cocktail APC 2.1 and expressed as “S” when susceptible or “R” when resistant (e.g., “is2S” means that isolate number 2 was susceptible to the phage cocktail) (Table 2 and Supplementary Methods). Among these eight isolates, analysis based on variant calling furthermore revealed four closely related but clearly delineated strains, i.e., clones that differ genetically from other clones, with different phage susceptibility phenotype (Table 2 and Figure 1): Strain 1 (Str1), contained isolates 1 and 3 (is1S and is3R), found in BAL#3 (is1S-Str1/BAL#3) and BAL#5 (is3R-Str1/BAL#5), respectively. Strain 2 (Str2) contained isolate 2 (is2S) from BAL#5 (is2S-Str2/BAL#5). Strain 3 (Str3) contained isolates 5, 6, and 9 (is5S, is6S and is9S), which were found in BAL#6 (is5S-Str3/BAL#6, is6S-Str3/BAL#6) and BAL#9 (is9S-Str3/BAL#9). Strain 4 contained isolates 7 and 8 (is7R and is8R), found in BAL#9 (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) (Table 2). Isolate is1S-Str1/BAL#3 was obtained on 25 July 2017, i.e., 45 days before the first phage therapy session (8 September 2017) and was 100% identical to is3R-Str1/BAL#5 isolated seven days (15 September 2017) after the first phage therapy session (Figure 1). Since both isolates is1S-Str1/BAL#3 and is3R-Str1/BAL#5 are 100% identical, this means that the phage resistance mechanism of isolate is3R-Str1/BAL#5 is not genetically supported (see discussion). Isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6, both phage susceptible, were obtained one month later (12 October 2017) and belonged to still another strain (Strain 3). Finally, on 8 February 2018, i.e., 16 days after the second phage therapy session (23 January 2018), an additional three isolates were obtained from BAL#9. Of these, isolate is9S-Str3/BAL#9 belonged to the same Strain 3 as isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6 and was phage susceptible as well, but isolates 7R and 8R belonged to still another strain (Strain 4) and were both resistant to the phage cocktail. Interestingly, the phage resistant Strain 4 isolates (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) contained mutations compared to the other six isolates that could explain the phage resistance observed for these two isolates (Figure 1): when Strain 1 was used as a reference, a C to A transition at position 1803 of the colicin I receptor gene caused a Tyr601->Stop transition. This receptor has been recognized as a phage receptor [7]. We further studied the course of infection by extracting DNA from the three available A. xylosoxidans-positive BAL samples (BAL#5, #6 and #9), and by quantifying DNA of the different strains by means of strain-specific qPCRs (Table 3 and Supplementary Methods) that had been developed on the basis of the obtained WGS data. qPCRs for Strain 2 and Strain 4 were only positive for the BALs from which these isolates had been cultured, respectively, BAL#5 and BAL#9. qPCR for Strain 1 was only positive for BAL#9, but not for BAL#5 from which isolate 3R had been cultured. qPCR for Strain 3 was positive for all three BALs, although no isolate belonging to Strain 3 was cultured from BAL#5. 3. Discussion and Conclusions We report the first case of decolonization of pandrug-resistant Achromobacter xylosoxidans after a long-term follow-up in a lung-transplanted patient receiving phage therapy. The first actual demonstration and usage of bacteriophages came almost simultaneously from two microbiologists in 1915 (Frederick Twort) and 1917 (Félix d’Hérelle) [8]. After extensive clinical use during the first half of the 20th century, their use has been progressively reduced, mainly due to the discovery, rapid spread, and ease of use of antibiotics for the treatment of infectious diseases [9]. However, bacteriophages gained a new surge of attention, due to the emergence and spread of pandrug-resistant bacteria [8]. Despite decades of clinical experience with bacteriophages in Eastern Europe, very few data are available regarding their clinical efficacy; besides, case-series and few randomized controlled trials (RCT) [10,11,12]. For instance, a RCT assessed the clinical outcome of patients receiving local application of a phage cocktail for the treatment of burn wound infection. This RCT is frequently cited to have shown no clinical benefits, as compared to standard of care. However, the phage titer that was applied (100 pfu/mL) [10], was so extremely low that it precludes to draw any conclusion from this study regarding the usefulness of phage therapy. The limited number of randomized clinical trials has been recently reviewed [13]. Phage therapy has also been used locally, e.g., for the treatment of bone and joint infections [14,15] or intravenously for the treatment of bloodstream infections [16,17]. Considering the high frequency of colonization and infection caused by antibiotic-resistant bacteria among cystic fibrosis and lung-transplanted patients, such as in our patient, the use of bacteriophages appears to be an appealing option in this population [18,19,20]. In the specific case of A. xylosoxidans, thus far a single case-report described the favorable outcome after the use of bacteriophages in a cystic fibrosis patient [21]. Our case-report highlights the many difficulties that need to be taken into account before considering the use of bacteriophages in a clinical setting. These include amongst others an important delay from clinical decision to regulatory agency authorization. Most problematic is the difficult interpretation of the clinical outcome after bacteriophage therapy, considering the long-term persisting airway colonization and the possible evolution of different A. xylosoxidans strains. The second therapeutic attempt relied on a massive lung instillation (injection of the bacteriophage solution in the patient’s lung during fibroscopy) followed by a two-week course of phage nebulization. We used a vibrating mesh nebulizer for phage delivery as this type of nebulizer has been shown to result in a lower phage titer reduction, as compared to jet nebulizers [22]. BAL#9, performed two weeks after the second round of phage therapy (8 February 2018) still grew 105 CFU/mL of A. xylosoxidans, represented by Strains 3 and 4 (according to culture) and by at least Strains 1, 3 and 4 (according to qPCR) (Table 1, Table 2 and Table 3). Sputum cultures remained positive for 18 months, but with only 103 CFU A. xylosoxidans/mL, suggesting a possible upper airway colonization, especially since no A. xylosoxidans could be isolated from BALs sampled in August 2019, and April 2020. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. To more precisely understand the discrepant outcome of our patient, another option would have been to search for active phages from the BAL samples throughout the study, but this was not performed. Despite the observed differences of a total of 367 SNPs between the four strains of eight A. xylosoxidans isolates from the patient, their overall similarity suggests that they originate from a single strain that colonized the patient (who was colonized with A. xylosoxidans years before the start of the phage therapy). Therefore, it is possible that some variants were present already before lung transplantation and before the start of phage therapy. Although it is not possible to exclude the simultaneous presence of these different strains on the basis of our culture results, because only one or two colonies were picked from each BAL culture, analysis by means of four strain-specific qPCR assays of the three BAL samples that were available for study, indicated that Strains 2 and 4 were probably prevalent at a particular moment of the course of infection, whereas Strains 1 and 3 could be identified during the full course of the infection. Phage resistant Strain 4 may have been selected during phage therapy. Phage training has been proposed to overcome phage resistance and increase infectivity against a given bacterial population [23]. However, training phages may take weeks, and therefore might be restricted to the treatment of chronic infections. Regarding more acute infections, the establishment of phage collections (such as the DSMZ, which provided bacteriophages for our patient) is of critical importance to provide phages active against a broad spectrum of bacterial isolates. An interesting point is the observation of two isolates from the same strain (is1S-Str1/BAL#3 and is3R-Str1/BAL#5) that differ regarding their phage susceptibility phenotype despite the absence of genetic support (identical genome). As observed with bacteria where emerging data point out that a resistance phenotype against a given antimicrobial may not be supported by a genotypic substratum [24,25], phage resistance without any change in genotype has also been documented [26]. These genotype/phenotype discrepancies are hypothesized to be possible through epigenetic regulation mechanisms, of which post-transcriptional regulation, which may influence phage receptor expression on the cell surface or phage receptor masking (through capsule synthesis). Further studies are required to better understand the dynamics of in vivo selection of phage-resistant bacteria. Because the patient’s medical condition improved anyhow, a few weeks after the second round of phage instillation, it is possible that phage therapy selected the phage-resistant A. xylosoxidans Strain 4, but that the mutation that caused phage resistance at the same time decreased the virulence of the strain as already described with other bacteria and phages [27,28,29]. Indeed, we observed several mutations which could explain the phage resistance of Strain 4, of which one was especially interesting, since it caused the gain of a stop codon in the colicin I receptor, which has been recognized as a phage receptor in other species [7]. Because in E. coli, the colicin I receptor plays a crucial role in iron transport across the outer membrane [7], loss of this function due to the observed mutation may have caused reduced fitness of the A. xylosoxidans mutant Strain 4. However, from a clinical point-of-view, one cannot exclude a spontaneous improvement, independently of phage therapy and despite months of fruitless antibiotic treatment, highlighting the dire need of randomized clinical trials in order to more convincingly assess the clinical efficacy of bacteriophages. In conclusion, we describe the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. Despite initial persisting airway colonization, the final clinical and microbiological outcome was favorable. Whole genome sequencing of eight A. xylosoxidans isolates and strain-specific qPCR experiments performed on BALs sampled before and after phage therapy allowed to more precisely study the dynamics of lung colonization by different isolates, susceptible or resistant to the applied phage cocktails. Acknowledgments The authors would like to thank L. Lécuyer and V. Boussaud (Unité de Transplantation, HEGP), B. Sabatier, V. Savoldelli, T. Caruba and P. Prognon (Pharmacie HEGP), P. Odou, C. Nassar, C. Berneron and M. Roche (Pharmacie Lille), and E. Deberranger (Hématologie Pédiatrique Lille), for their help in the management of the patient. Microbiological analysis was performed with a funding by the Association Robert Debré pour la Recherche Médicale. The authors would also like to thank Caroline Semaille and Nathalie Morgensztejn from the Agence Nationale de Sécurité du Médicament (ANSM) for their regulatory support. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Materials The following are available online at https://www.mdpi.com/1999-4915/13/1/60/s1, Figure S1: Antibiotic susceptibility testing (AST) of a pandrug-resistant Achromobacter xylosoxidans isolated during a bronchoalveolar lavage of a 12-year-old boy after double lung transplantation, Table S1: Variant calling for the eight Achromobacter xylosoxidans whole genome sequences, see excel file attached, named: “Supplementary Table S1. Variant calling.xls”. Click here for additional data file. Author Contributions D.L. (David Lebeaux) and M.V. conceptualized the study, validated the data, carried out formal analysis, prepared the initial draft, took care of project administration and supervised the study. Data were visualized by H.D. and Y.G. Software analysis was carried out by H.D., S.V., Y.G. D.L. (David Lebeaux), M.M., R.G., I.P., F.C., N.K., J.W., C.R., E.C., D.L. (Damien Lanoy), L.V.S., H.D., J.-P.P., N.D., S.V., Y.G., F.V.N., and M.V. contributed to basic methodology. Data curation was done by D.L. (David Lebeaux), M.M., C.T., S.V., Y.G., and M.V. Funding was acquired by D.L. (David Lebeaux), J.-L.M., F.V.N., and M.V. All authors took part in the investigation and in final reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement The study did not require ethical approval. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Phylogenetic tree built with Whole genome sequences of eight Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. The distance between samples reflects the number of different Single Nucleotide Polymorphisms (SNPs). Each A. xylosoxidans is named as follows: isolate number (is1 to is8) S (or R to depict susceptibility or resistance to phage cocktail)-Strain number/Number of BAL where it has been identified. viruses-13-00060-t001_Table 1Table 1 Results of bronchoalveolar lavages (BALs) and sputum analysis from May 2017, to April 2020, in 12-year-old lung transplanted patient who received two rounds of phage therapy for pandrug-resistant Achromobacter xylosoxidans colonization and infection. Date 19 May 2017 23 May 2017 6 June 2017 12 June 2017 25 July 2017 5 September 2017 8 September 2017 15 September 2017 12 October 2017 27 October 2017 23 January 2018 8 February 2018 5 March 2018 18 May 2018 19 February 2019 19 June 2019 27 August 2019 30 April 2020 Phage therapy ROUND 1 ROUND 2 Tigecycline from 29 June to 31 July Imipenem From 31 July 2017, to 16 February 2018 Number of days, relative to the first round of phage therapy −112 −108 −94 −88 −45 −3 +7 +37 +49 +137 +153 +178 +252 +529 +649 +718 +963 BAL BAL Number * #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 Leukocytes (/mm3) >1000 800 >1000 500 >1000 60 >1000 810 >1000 NA NA NA PMN cells (%) 90 88 90 91 43 30 80 69 82 NA NA NA Quantification of A. x culture (CFU/mL) 104 103 104 103 104 103 104 106 105 104 0 0 Other bacteria (CFU/mL) None None None None None None None None None None 104 S. a 106 S. a Sputum Leucocytes (/field) >25 >25 >25 >25 NA NA NA Quantification of A. x culture (CFU/mL) 105 105 105 106 103 103 103 Other bacteria (CFU/mL) None None None None 108 M. c None 106 S. a A. x: Achromobacter xylosoxidans; M. c: Moraxella catarrhalis; NA: non available; PMN: Polymorphonuclear; RBC: red blood cells; S. a: Staphylococcus aureus. : BAL number in bold indicates BALs for which qPCR had been carried out. viruses-13-00060-t002_Table 2Table 2 Eight Achromobacter xylosoxidans isolates for which whole genome sequencing was carried out. These were isolated from bronchoalveolar lavages in a 12-year-old boy after double lung transplantation. The patient received a first round of phage administration on 8 September 2017. A second round of phage treatment was performed on 23 January 2018. Complete Name Isolate Strain Source of Sampling Date of Sampling Whole Genome Sequence is1S-Str1/BAL#3 is1S Str1 BAL#3 25 July 2017 ERS5044236-UGAX1 is2S-Str2/BAL#5 is2S Str2 BAL#5 15 September 2017 ERS5044237-UGAX2 is3R-Str1/BAL#5 is3R Str1 BAL#5 15 September 2017 ERS5044238-UGAX3 is5S-Str3/BAL#6 is5S Str3 BAL#6 12 October 2017 ERS5044239-UGAX5 is6S-Str3/BAL#6 is6S Str3 BAL#6 12 October 2017 ERS5044240-UGAX6 is7R-Str4/BAL#9 is7R Str4 BAL#9 8 February 2018 ERS5044241-UGAX7 is8R-Str4/BAL#9 is8R Str4 BAL#9 8 February 2018 ERS5044242-UGAX8 is9S-Str3/BAL#9 is9S Str3 BAL#9 8 February 2018 ERS5044243-UGAX9 * Submitted at ENA, 1 September 2020. Study ID: PRJEB39103. BAL: Bronchoalveolar lavage, S: Phage susceptible, R: phage resistant. viruses-13-00060-t003_Table 3Table 3 Results of bacterial culture (grey-shading) and qPCR of three broncho-alveolar lavages for four different Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. Bronchoalveolar Lavage BAL#5 BAL#6 BAL#9 15 September 2017 12 October 2017 8 February 2018 qPCR for Strain Isolates Str1 is1S, is3R N NS P (34.7) Str2 is2S P (36.6) N N Str3 is5S, is6S, is9S P (32.7) P (28.9) P (27.7) Str4 is7R, is8R NS NS P (35.2) Grey-shaded box = isolate cultured from this BAL; N: negative (Cq-value > 40); NS: Nonspecific amplification (melting peak different from that of specific amplification); P: positive qPCR (Cq-value).	25 mg (milligrams).	DrugDosage	CC BY	33466377	19,957,163	2021-01-05
What was the dosage of drug 'SULFAMETHOXAZOLE\TRIMETHOPRIM'?	A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient. Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains. 1. Case Presentation We report the case of a 12-year-old boy (35.5 kg), who received double lung transplantation in 25 March 2017, (at the age of 12) because of cystic fibrosis. Before transplantation, he was colonized with Aspergillus fumigatus, Achromobacter xylosoxidans (first colonization identified in 2013), and Pseudomonas aeruginosa. After transplantation, he experienced acute kidney injury requiring temporary haemodialysis, pulmonary embolism, and persisting airway colonization with A. fumigatus. He was discharged on 29 April 2017, with satisfactory respiratory status; he initially required no home oxygen therapy. His immunosuppressive regimen included prednisolone (25 mg/day), tacrolimus (1.5 mg bis in die (b.i.d.) with a target trough concentration of 6–8 ng/mL), mycophenolate mofetil (1 g b.i.d.) and his antimicrobial prophylaxis was cotrimoxazole (400/80 mg, three times a week), posaconazole (150 mg, oral suspension, ter in die (t.i.d.)), valacyclovir (500 mg b.i.d.), and intravenous immunoglobulins (15 g each three weeks). Between May and June 2017, he experienced progressive shortness of breath, cough and increased sputum production; he subsequently required the addition of 1–1.5 L/min oxygen therapy. Because of a stenosis of the surgical anastomosis of the main left bronchus, a dilatation during rigid bronchoscopy was performed on 19 May and a Montgomery stent was inserted on 2 June. Later on, a stenosis of the surgical anastomosis on the right bronchus intermedius required a dilatation during rigid bronchoscopy followed by the insertion of an Oki stent on 29 June. Several bronchoalveolar lavages (BAL), performed between May and June 2017, revealed inflammation (between 800 and >1000 leukocytes/mm3 with 88–90% of polymorphonuclear cells) and repeatedly grew pandrug-resistant A. xylosoxidans (Table 1 and Supplementary Figure S1) [1]. Lung biopsy performed on 19 June was consistent with lung infection: diffuse acute bronchiolitis with extension to peri-bronchiolar alveoli. Microbiological samples obtained from lung biopsies were negative (bacterial, mycological, and mycobacteriological cultures). The first antibiotic treatment consisted of intravenous tigecycline 50 mg b.i.d. from 29 June to 31 July. As BAL remained positive for A. xylosoxidans and the patient still required oxygen therapy; antibiotic treatment was subsequently switched to imipenem (1200 mg, t.i.d., ~100 mg/Kg/d) on 31 July 2017. Subsequent respiratory samples still grew A. xylosoxidans and the respiratory status did not improve (requiring oxygen at home, 1 L/min). Furthermore, despite antibiotic treatment and correct drainage of the right superior lobe, lung consolidation and micronodules remained. Considering the failure of antibiotic treatment, phage therapy was proposed to the patient and his family, after multidisciplinary discussion. A first cocktail (APC 1.1) containing three lytic phages (JWDelta, JWT and 2-1) active against A. xylosoxidans isolate is1S (designated such as later), selected from the DSMZ collection (Braunschweig, Germany) [2,3], was prepared at the Laboratory for Bacteriology Research (Ghent University, Belgium) and the Laboratory for Molecular and Cellular Technology (Queen Astrid Military Hospital, Brussels, Belgium) using the above-mentioned bacterial isolate, as previously described [4]. The production was completed on 23 August with a final phage titer of 4 × 1010 plaque forming units (pfu)/mL defined by double-agar overlay method [4], an endotoxin level of 4000 EU/mL (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, GenScript, Piscataway Township, NJ, USA) and a pH of 7.3 (in sterile PBS). Regulatory permission for phage importation was obtained on 1 September 2017, from the Agence Nationale de Sécurité du Médicament (ANSM). A first round of phage administration was performed, consisting of 3 nebulizations/day of 5 mL of the filter-sterilized (Sartorius 0.2 µm) phage solution, tenfold diluted in sterile saline on 8 and 9 September, using a vibrating mesh nebulizer (eFLOW rapid, PARI, PARI GmbH, Germany) with a mouth piece. Immediate tolerance was perfect, but no clinical improvement was noted and culture of subsequent respiratory samples remained positive for A. xylosoxidans (104 CFU/mL in the BAL of 15 September). A second cocktail (APC 2.1) was produced, in which phage JWalpha was added to the three lytic phages of cocktail APC 1.1, to improve the therapeutic efficacy. The production was completed on 22 December with a final phage titer of 5 × 109 pfu/mL, an endotoxin level of 1760 EU/mL and a pH of 6.95 (in sterile PBS). Regulatory permission was obtained for phage importation on 16 January 2018 (ANSM). On 23 January, during therapeutic bronchoscopy under general anesthesia, 30 mL of APC2.1, tenfold diluted (in sterile saline) and filter-sterilized, was instilled in each pulmonary lobe through the fibroscope. Immediate tolerance was perfect and the patient was discharged on 24 January with continued phage nebulization at home: three times a day 5 mL of preparation A using a vibrating mesh nebulizer (eFLOW rapid, PARI) with a mouth piece for 14 days until 6 February. Clinical tolerance was perfect again, but the initial clinical status remained unchanged. BAL performed on 8 February 2018, two days after stopping the phage treatment, still grew 105 cfu/mL of A. xylosoxidans. Subsequently, the patient’s respiratory condition slowly improved and oxygen therapy was stopped on 15 February 2018. No clinical worsening was observed after imipenem interruption on 16 February 2018. Sputum culture remained positive for A. xylosoxidans but with a low bacterial density (103 CFU/mL in March and May 2018, and June 2019) and no A. xylosoxidans could be isolated from BALs#10 and #11, sampled on August 2019, and April 2020, respectively (Table 1). The last pulmonary function tests, performed on 17 October 2019, showed best results since lung transplantation, with an FEV1 of 79% and a forced vital capacity of 85%. 2. Microbiological Analysis of Eight A. xylosoxidans Isolates As we observed discrepant outcome in our patient (clinical improvement on the one hand but persisting low-grade airway colonization on the other hand), we decided to study the eight available A. xylosoxidans isolates (is1 to is9) from the patient’s colonization obtained by random picking from the culture plates from 4 distinct BALs (Table 2). These eight isolates, taken before, during and after the phage therapy period (Table 2), could not be differentiated from each other by means of MALDI-TOF [5] or McRAPD typing [6] and all belonged to the epidemic clone, already described previously among Belgian CF patients [5]. Whole genome sequencing (WGS) and de novo genome assembly for these eight isolates was carried out (Supplementary Methods and Table S1). The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Total genome length for all eight isolates was 6.44–6.50 Mbp, with a GC content of 67.5%. All eight isolates showed most similarity with reference genome GCF_008432465 (AX1), which corresponds to the type strain of A. xylosoxidans subsp. xylosoxidans (ATCC 27061T, CCUG 12689T, DSMZ 10346T, LMG 1863T, and NCTC 10807T), i.e., between 88.38 and 89.60% on the basis of mapped sequence reads, and between 98.71% and 98.76% on the basis of average nucleotide identity (ANI). Indeed, the overall similarity between the genome sequences of the eight isolates was high, i.e., 99.60%, as assessed by digital DNA hybridization, and more than 99.97% according to ANI-analysis. This was confirmed by variant calling where we observed that the isolates differed from each other only for a total of 367 SNPs, whereas a total of 59,452 SNPs were observed between the isolates and the best matching reference genome (AX1) (Figure 1). The susceptibility phenotype of each of the eight isolates was assessed by spot-test against the phage cocktail APC 2.1 and expressed as “S” when susceptible or “R” when resistant (e.g., “is2S” means that isolate number 2 was susceptible to the phage cocktail) (Table 2 and Supplementary Methods). Among these eight isolates, analysis based on variant calling furthermore revealed four closely related but clearly delineated strains, i.e., clones that differ genetically from other clones, with different phage susceptibility phenotype (Table 2 and Figure 1): Strain 1 (Str1), contained isolates 1 and 3 (is1S and is3R), found in BAL#3 (is1S-Str1/BAL#3) and BAL#5 (is3R-Str1/BAL#5), respectively. Strain 2 (Str2) contained isolate 2 (is2S) from BAL#5 (is2S-Str2/BAL#5). Strain 3 (Str3) contained isolates 5, 6, and 9 (is5S, is6S and is9S), which were found in BAL#6 (is5S-Str3/BAL#6, is6S-Str3/BAL#6) and BAL#9 (is9S-Str3/BAL#9). Strain 4 contained isolates 7 and 8 (is7R and is8R), found in BAL#9 (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) (Table 2). Isolate is1S-Str1/BAL#3 was obtained on 25 July 2017, i.e., 45 days before the first phage therapy session (8 September 2017) and was 100% identical to is3R-Str1/BAL#5 isolated seven days (15 September 2017) after the first phage therapy session (Figure 1). Since both isolates is1S-Str1/BAL#3 and is3R-Str1/BAL#5 are 100% identical, this means that the phage resistance mechanism of isolate is3R-Str1/BAL#5 is not genetically supported (see discussion). Isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6, both phage susceptible, were obtained one month later (12 October 2017) and belonged to still another strain (Strain 3). Finally, on 8 February 2018, i.e., 16 days after the second phage therapy session (23 January 2018), an additional three isolates were obtained from BAL#9. Of these, isolate is9S-Str3/BAL#9 belonged to the same Strain 3 as isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6 and was phage susceptible as well, but isolates 7R and 8R belonged to still another strain (Strain 4) and were both resistant to the phage cocktail. Interestingly, the phage resistant Strain 4 isolates (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) contained mutations compared to the other six isolates that could explain the phage resistance observed for these two isolates (Figure 1): when Strain 1 was used as a reference, a C to A transition at position 1803 of the colicin I receptor gene caused a Tyr601->Stop transition. This receptor has been recognized as a phage receptor [7]. We further studied the course of infection by extracting DNA from the three available A. xylosoxidans-positive BAL samples (BAL#5, #6 and #9), and by quantifying DNA of the different strains by means of strain-specific qPCRs (Table 3 and Supplementary Methods) that had been developed on the basis of the obtained WGS data. qPCRs for Strain 2 and Strain 4 were only positive for the BALs from which these isolates had been cultured, respectively, BAL#5 and BAL#9. qPCR for Strain 1 was only positive for BAL#9, but not for BAL#5 from which isolate 3R had been cultured. qPCR for Strain 3 was positive for all three BALs, although no isolate belonging to Strain 3 was cultured from BAL#5. 3. Discussion and Conclusions We report the first case of decolonization of pandrug-resistant Achromobacter xylosoxidans after a long-term follow-up in a lung-transplanted patient receiving phage therapy. The first actual demonstration and usage of bacteriophages came almost simultaneously from two microbiologists in 1915 (Frederick Twort) and 1917 (Félix d’Hérelle) [8]. After extensive clinical use during the first half of the 20th century, their use has been progressively reduced, mainly due to the discovery, rapid spread, and ease of use of antibiotics for the treatment of infectious diseases [9]. However, bacteriophages gained a new surge of attention, due to the emergence and spread of pandrug-resistant bacteria [8]. Despite decades of clinical experience with bacteriophages in Eastern Europe, very few data are available regarding their clinical efficacy; besides, case-series and few randomized controlled trials (RCT) [10,11,12]. For instance, a RCT assessed the clinical outcome of patients receiving local application of a phage cocktail for the treatment of burn wound infection. This RCT is frequently cited to have shown no clinical benefits, as compared to standard of care. However, the phage titer that was applied (100 pfu/mL) [10], was so extremely low that it precludes to draw any conclusion from this study regarding the usefulness of phage therapy. The limited number of randomized clinical trials has been recently reviewed [13]. Phage therapy has also been used locally, e.g., for the treatment of bone and joint infections [14,15] or intravenously for the treatment of bloodstream infections [16,17]. Considering the high frequency of colonization and infection caused by antibiotic-resistant bacteria among cystic fibrosis and lung-transplanted patients, such as in our patient, the use of bacteriophages appears to be an appealing option in this population [18,19,20]. In the specific case of A. xylosoxidans, thus far a single case-report described the favorable outcome after the use of bacteriophages in a cystic fibrosis patient [21]. Our case-report highlights the many difficulties that need to be taken into account before considering the use of bacteriophages in a clinical setting. These include amongst others an important delay from clinical decision to regulatory agency authorization. Most problematic is the difficult interpretation of the clinical outcome after bacteriophage therapy, considering the long-term persisting airway colonization and the possible evolution of different A. xylosoxidans strains. The second therapeutic attempt relied on a massive lung instillation (injection of the bacteriophage solution in the patient’s lung during fibroscopy) followed by a two-week course of phage nebulization. We used a vibrating mesh nebulizer for phage delivery as this type of nebulizer has been shown to result in a lower phage titer reduction, as compared to jet nebulizers [22]. BAL#9, performed two weeks after the second round of phage therapy (8 February 2018) still grew 105 CFU/mL of A. xylosoxidans, represented by Strains 3 and 4 (according to culture) and by at least Strains 1, 3 and 4 (according to qPCR) (Table 1, Table 2 and Table 3). Sputum cultures remained positive for 18 months, but with only 103 CFU A. xylosoxidans/mL, suggesting a possible upper airway colonization, especially since no A. xylosoxidans could be isolated from BALs sampled in August 2019, and April 2020. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. To more precisely understand the discrepant outcome of our patient, another option would have been to search for active phages from the BAL samples throughout the study, but this was not performed. Despite the observed differences of a total of 367 SNPs between the four strains of eight A. xylosoxidans isolates from the patient, their overall similarity suggests that they originate from a single strain that colonized the patient (who was colonized with A. xylosoxidans years before the start of the phage therapy). Therefore, it is possible that some variants were present already before lung transplantation and before the start of phage therapy. Although it is not possible to exclude the simultaneous presence of these different strains on the basis of our culture results, because only one or two colonies were picked from each BAL culture, analysis by means of four strain-specific qPCR assays of the three BAL samples that were available for study, indicated that Strains 2 and 4 were probably prevalent at a particular moment of the course of infection, whereas Strains 1 and 3 could be identified during the full course of the infection. Phage resistant Strain 4 may have been selected during phage therapy. Phage training has been proposed to overcome phage resistance and increase infectivity against a given bacterial population [23]. However, training phages may take weeks, and therefore might be restricted to the treatment of chronic infections. Regarding more acute infections, the establishment of phage collections (such as the DSMZ, which provided bacteriophages for our patient) is of critical importance to provide phages active against a broad spectrum of bacterial isolates. An interesting point is the observation of two isolates from the same strain (is1S-Str1/BAL#3 and is3R-Str1/BAL#5) that differ regarding their phage susceptibility phenotype despite the absence of genetic support (identical genome). As observed with bacteria where emerging data point out that a resistance phenotype against a given antimicrobial may not be supported by a genotypic substratum [24,25], phage resistance without any change in genotype has also been documented [26]. These genotype/phenotype discrepancies are hypothesized to be possible through epigenetic regulation mechanisms, of which post-transcriptional regulation, which may influence phage receptor expression on the cell surface or phage receptor masking (through capsule synthesis). Further studies are required to better understand the dynamics of in vivo selection of phage-resistant bacteria. Because the patient’s medical condition improved anyhow, a few weeks after the second round of phage instillation, it is possible that phage therapy selected the phage-resistant A. xylosoxidans Strain 4, but that the mutation that caused phage resistance at the same time decreased the virulence of the strain as already described with other bacteria and phages [27,28,29]. Indeed, we observed several mutations which could explain the phage resistance of Strain 4, of which one was especially interesting, since it caused the gain of a stop codon in the colicin I receptor, which has been recognized as a phage receptor in other species [7]. Because in E. coli, the colicin I receptor plays a crucial role in iron transport across the outer membrane [7], loss of this function due to the observed mutation may have caused reduced fitness of the A. xylosoxidans mutant Strain 4. However, from a clinical point-of-view, one cannot exclude a spontaneous improvement, independently of phage therapy and despite months of fruitless antibiotic treatment, highlighting the dire need of randomized clinical trials in order to more convincingly assess the clinical efficacy of bacteriophages. In conclusion, we describe the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. Despite initial persisting airway colonization, the final clinical and microbiological outcome was favorable. Whole genome sequencing of eight A. xylosoxidans isolates and strain-specific qPCR experiments performed on BALs sampled before and after phage therapy allowed to more precisely study the dynamics of lung colonization by different isolates, susceptible or resistant to the applied phage cocktails. Acknowledgments The authors would like to thank L. Lécuyer and V. Boussaud (Unité de Transplantation, HEGP), B. Sabatier, V. Savoldelli, T. Caruba and P. Prognon (Pharmacie HEGP), P. Odou, C. Nassar, C. Berneron and M. Roche (Pharmacie Lille), and E. Deberranger (Hématologie Pédiatrique Lille), for their help in the management of the patient. Microbiological analysis was performed with a funding by the Association Robert Debré pour la Recherche Médicale. The authors would also like to thank Caroline Semaille and Nathalie Morgensztejn from the Agence Nationale de Sécurité du Médicament (ANSM) for their regulatory support. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Materials The following are available online at https://www.mdpi.com/1999-4915/13/1/60/s1, Figure S1: Antibiotic susceptibility testing (AST) of a pandrug-resistant Achromobacter xylosoxidans isolated during a bronchoalveolar lavage of a 12-year-old boy after double lung transplantation, Table S1: Variant calling for the eight Achromobacter xylosoxidans whole genome sequences, see excel file attached, named: “Supplementary Table S1. Variant calling.xls”. Click here for additional data file. Author Contributions D.L. (David Lebeaux) and M.V. conceptualized the study, validated the data, carried out formal analysis, prepared the initial draft, took care of project administration and supervised the study. Data were visualized by H.D. and Y.G. Software analysis was carried out by H.D., S.V., Y.G. D.L. (David Lebeaux), M.M., R.G., I.P., F.C., N.K., J.W., C.R., E.C., D.L. (Damien Lanoy), L.V.S., H.D., J.-P.P., N.D., S.V., Y.G., F.V.N., and M.V. contributed to basic methodology. Data curation was done by D.L. (David Lebeaux), M.M., C.T., S.V., Y.G., and M.V. Funding was acquired by D.L. (David Lebeaux), J.-L.M., F.V.N., and M.V. All authors took part in the investigation and in final reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement The study did not require ethical approval. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Phylogenetic tree built with Whole genome sequences of eight Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. The distance between samples reflects the number of different Single Nucleotide Polymorphisms (SNPs). Each A. xylosoxidans is named as follows: isolate number (is1 to is8) S (or R to depict susceptibility or resistance to phage cocktail)-Strain number/Number of BAL where it has been identified. viruses-13-00060-t001_Table 1Table 1 Results of bronchoalveolar lavages (BALs) and sputum analysis from May 2017, to April 2020, in 12-year-old lung transplanted patient who received two rounds of phage therapy for pandrug-resistant Achromobacter xylosoxidans colonization and infection. Date 19 May 2017 23 May 2017 6 June 2017 12 June 2017 25 July 2017 5 September 2017 8 September 2017 15 September 2017 12 October 2017 27 October 2017 23 January 2018 8 February 2018 5 March 2018 18 May 2018 19 February 2019 19 June 2019 27 August 2019 30 April 2020 Phage therapy ROUND 1 ROUND 2 Tigecycline from 29 June to 31 July Imipenem From 31 July 2017, to 16 February 2018 Number of days, relative to the first round of phage therapy −112 −108 −94 −88 −45 −3 +7 +37 +49 +137 +153 +178 +252 +529 +649 +718 +963 BAL BAL Number * #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 Leukocytes (/mm3) >1000 800 >1000 500 >1000 60 >1000 810 >1000 NA NA NA PMN cells (%) 90 88 90 91 43 30 80 69 82 NA NA NA Quantification of A. x culture (CFU/mL) 104 103 104 103 104 103 104 106 105 104 0 0 Other bacteria (CFU/mL) None None None None None None None None None None 104 S. a 106 S. a Sputum Leucocytes (/field) >25 >25 >25 >25 NA NA NA Quantification of A. x culture (CFU/mL) 105 105 105 106 103 103 103 Other bacteria (CFU/mL) None None None None 108 M. c None 106 S. a A. x: Achromobacter xylosoxidans; M. c: Moraxella catarrhalis; NA: non available; PMN: Polymorphonuclear; RBC: red blood cells; S. a: Staphylococcus aureus. : BAL number in bold indicates BALs for which qPCR had been carried out. viruses-13-00060-t002_Table 2Table 2 Eight Achromobacter xylosoxidans isolates for which whole genome sequencing was carried out. These were isolated from bronchoalveolar lavages in a 12-year-old boy after double lung transplantation. The patient received a first round of phage administration on 8 September 2017. A second round of phage treatment was performed on 23 January 2018. Complete Name Isolate Strain Source of Sampling Date of Sampling Whole Genome Sequence is1S-Str1/BAL#3 is1S Str1 BAL#3 25 July 2017 ERS5044236-UGAX1 is2S-Str2/BAL#5 is2S Str2 BAL#5 15 September 2017 ERS5044237-UGAX2 is3R-Str1/BAL#5 is3R Str1 BAL#5 15 September 2017 ERS5044238-UGAX3 is5S-Str3/BAL#6 is5S Str3 BAL#6 12 October 2017 ERS5044239-UGAX5 is6S-Str3/BAL#6 is6S Str3 BAL#6 12 October 2017 ERS5044240-UGAX6 is7R-Str4/BAL#9 is7R Str4 BAL#9 8 February 2018 ERS5044241-UGAX7 is8R-Str4/BAL#9 is8R Str4 BAL#9 8 February 2018 ERS5044242-UGAX8 is9S-Str3/BAL#9 is9S Str3 BAL#9 8 February 2018 ERS5044243-UGAX9 * Submitted at ENA, 1 September 2020. Study ID: PRJEB39103. BAL: Bronchoalveolar lavage, S: Phage susceptible, R: phage resistant. viruses-13-00060-t003_Table 3Table 3 Results of bacterial culture (grey-shading) and qPCR of three broncho-alveolar lavages for four different Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. Bronchoalveolar Lavage BAL#5 BAL#6 BAL#9 15 September 2017 12 October 2017 8 February 2018 qPCR for Strain Isolates Str1 is1S, is3R N NS P (34.7) Str2 is2S P (36.6) N N Str3 is5S, is6S, is9S P (32.7) P (28.9) P (27.7) Str4 is7R, is8R NS NS P (35.2) Grey-shaded box = isolate cultured from this BAL; N: negative (Cq-value > 40); NS: Nonspecific amplification (melting peak different from that of specific amplification); P: positive qPCR (Cq-value).	400/80 MG, THRICE WEEKLY	DrugDosageText	CC BY	33466377	19,957,163	2021-01-05
What was the dosage of drug 'TACROLIMUS'?	A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient. Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains. 1. Case Presentation We report the case of a 12-year-old boy (35.5 kg), who received double lung transplantation in 25 March 2017, (at the age of 12) because of cystic fibrosis. Before transplantation, he was colonized with Aspergillus fumigatus, Achromobacter xylosoxidans (first colonization identified in 2013), and Pseudomonas aeruginosa. After transplantation, he experienced acute kidney injury requiring temporary haemodialysis, pulmonary embolism, and persisting airway colonization with A. fumigatus. He was discharged on 29 April 2017, with satisfactory respiratory status; he initially required no home oxygen therapy. His immunosuppressive regimen included prednisolone (25 mg/day), tacrolimus (1.5 mg bis in die (b.i.d.) with a target trough concentration of 6–8 ng/mL), mycophenolate mofetil (1 g b.i.d.) and his antimicrobial prophylaxis was cotrimoxazole (400/80 mg, three times a week), posaconazole (150 mg, oral suspension, ter in die (t.i.d.)), valacyclovir (500 mg b.i.d.), and intravenous immunoglobulins (15 g each three weeks). Between May and June 2017, he experienced progressive shortness of breath, cough and increased sputum production; he subsequently required the addition of 1–1.5 L/min oxygen therapy. Because of a stenosis of the surgical anastomosis of the main left bronchus, a dilatation during rigid bronchoscopy was performed on 19 May and a Montgomery stent was inserted on 2 June. Later on, a stenosis of the surgical anastomosis on the right bronchus intermedius required a dilatation during rigid bronchoscopy followed by the insertion of an Oki stent on 29 June. Several bronchoalveolar lavages (BAL), performed between May and June 2017, revealed inflammation (between 800 and >1000 leukocytes/mm3 with 88–90% of polymorphonuclear cells) and repeatedly grew pandrug-resistant A. xylosoxidans (Table 1 and Supplementary Figure S1) [1]. Lung biopsy performed on 19 June was consistent with lung infection: diffuse acute bronchiolitis with extension to peri-bronchiolar alveoli. Microbiological samples obtained from lung biopsies were negative (bacterial, mycological, and mycobacteriological cultures). The first antibiotic treatment consisted of intravenous tigecycline 50 mg b.i.d. from 29 June to 31 July. As BAL remained positive for A. xylosoxidans and the patient still required oxygen therapy; antibiotic treatment was subsequently switched to imipenem (1200 mg, t.i.d., ~100 mg/Kg/d) on 31 July 2017. Subsequent respiratory samples still grew A. xylosoxidans and the respiratory status did not improve (requiring oxygen at home, 1 L/min). Furthermore, despite antibiotic treatment and correct drainage of the right superior lobe, lung consolidation and micronodules remained. Considering the failure of antibiotic treatment, phage therapy was proposed to the patient and his family, after multidisciplinary discussion. A first cocktail (APC 1.1) containing three lytic phages (JWDelta, JWT and 2-1) active against A. xylosoxidans isolate is1S (designated such as later), selected from the DSMZ collection (Braunschweig, Germany) [2,3], was prepared at the Laboratory for Bacteriology Research (Ghent University, Belgium) and the Laboratory for Molecular and Cellular Technology (Queen Astrid Military Hospital, Brussels, Belgium) using the above-mentioned bacterial isolate, as previously described [4]. The production was completed on 23 August with a final phage titer of 4 × 1010 plaque forming units (pfu)/mL defined by double-agar overlay method [4], an endotoxin level of 4000 EU/mL (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, GenScript, Piscataway Township, NJ, USA) and a pH of 7.3 (in sterile PBS). Regulatory permission for phage importation was obtained on 1 September 2017, from the Agence Nationale de Sécurité du Médicament (ANSM). A first round of phage administration was performed, consisting of 3 nebulizations/day of 5 mL of the filter-sterilized (Sartorius 0.2 µm) phage solution, tenfold diluted in sterile saline on 8 and 9 September, using a vibrating mesh nebulizer (eFLOW rapid, PARI, PARI GmbH, Germany) with a mouth piece. Immediate tolerance was perfect, but no clinical improvement was noted and culture of subsequent respiratory samples remained positive for A. xylosoxidans (104 CFU/mL in the BAL of 15 September). A second cocktail (APC 2.1) was produced, in which phage JWalpha was added to the three lytic phages of cocktail APC 1.1, to improve the therapeutic efficacy. The production was completed on 22 December with a final phage titer of 5 × 109 pfu/mL, an endotoxin level of 1760 EU/mL and a pH of 6.95 (in sterile PBS). Regulatory permission was obtained for phage importation on 16 January 2018 (ANSM). On 23 January, during therapeutic bronchoscopy under general anesthesia, 30 mL of APC2.1, tenfold diluted (in sterile saline) and filter-sterilized, was instilled in each pulmonary lobe through the fibroscope. Immediate tolerance was perfect and the patient was discharged on 24 January with continued phage nebulization at home: three times a day 5 mL of preparation A using a vibrating mesh nebulizer (eFLOW rapid, PARI) with a mouth piece for 14 days until 6 February. Clinical tolerance was perfect again, but the initial clinical status remained unchanged. BAL performed on 8 February 2018, two days after stopping the phage treatment, still grew 105 cfu/mL of A. xylosoxidans. Subsequently, the patient’s respiratory condition slowly improved and oxygen therapy was stopped on 15 February 2018. No clinical worsening was observed after imipenem interruption on 16 February 2018. Sputum culture remained positive for A. xylosoxidans but with a low bacterial density (103 CFU/mL in March and May 2018, and June 2019) and no A. xylosoxidans could be isolated from BALs#10 and #11, sampled on August 2019, and April 2020, respectively (Table 1). The last pulmonary function tests, performed on 17 October 2019, showed best results since lung transplantation, with an FEV1 of 79% and a forced vital capacity of 85%. 2. Microbiological Analysis of Eight A. xylosoxidans Isolates As we observed discrepant outcome in our patient (clinical improvement on the one hand but persisting low-grade airway colonization on the other hand), we decided to study the eight available A. xylosoxidans isolates (is1 to is9) from the patient’s colonization obtained by random picking from the culture plates from 4 distinct BALs (Table 2). These eight isolates, taken before, during and after the phage therapy period (Table 2), could not be differentiated from each other by means of MALDI-TOF [5] or McRAPD typing [6] and all belonged to the epidemic clone, already described previously among Belgian CF patients [5]. Whole genome sequencing (WGS) and de novo genome assembly for these eight isolates was carried out (Supplementary Methods and Table S1). The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Total genome length for all eight isolates was 6.44–6.50 Mbp, with a GC content of 67.5%. All eight isolates showed most similarity with reference genome GCF_008432465 (AX1), which corresponds to the type strain of A. xylosoxidans subsp. xylosoxidans (ATCC 27061T, CCUG 12689T, DSMZ 10346T, LMG 1863T, and NCTC 10807T), i.e., between 88.38 and 89.60% on the basis of mapped sequence reads, and between 98.71% and 98.76% on the basis of average nucleotide identity (ANI). Indeed, the overall similarity between the genome sequences of the eight isolates was high, i.e., 99.60%, as assessed by digital DNA hybridization, and more than 99.97% according to ANI-analysis. This was confirmed by variant calling where we observed that the isolates differed from each other only for a total of 367 SNPs, whereas a total of 59,452 SNPs were observed between the isolates and the best matching reference genome (AX1) (Figure 1). The susceptibility phenotype of each of the eight isolates was assessed by spot-test against the phage cocktail APC 2.1 and expressed as “S” when susceptible or “R” when resistant (e.g., “is2S” means that isolate number 2 was susceptible to the phage cocktail) (Table 2 and Supplementary Methods). Among these eight isolates, analysis based on variant calling furthermore revealed four closely related but clearly delineated strains, i.e., clones that differ genetically from other clones, with different phage susceptibility phenotype (Table 2 and Figure 1): Strain 1 (Str1), contained isolates 1 and 3 (is1S and is3R), found in BAL#3 (is1S-Str1/BAL#3) and BAL#5 (is3R-Str1/BAL#5), respectively. Strain 2 (Str2) contained isolate 2 (is2S) from BAL#5 (is2S-Str2/BAL#5). Strain 3 (Str3) contained isolates 5, 6, and 9 (is5S, is6S and is9S), which were found in BAL#6 (is5S-Str3/BAL#6, is6S-Str3/BAL#6) and BAL#9 (is9S-Str3/BAL#9). Strain 4 contained isolates 7 and 8 (is7R and is8R), found in BAL#9 (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) (Table 2). Isolate is1S-Str1/BAL#3 was obtained on 25 July 2017, i.e., 45 days before the first phage therapy session (8 September 2017) and was 100% identical to is3R-Str1/BAL#5 isolated seven days (15 September 2017) after the first phage therapy session (Figure 1). Since both isolates is1S-Str1/BAL#3 and is3R-Str1/BAL#5 are 100% identical, this means that the phage resistance mechanism of isolate is3R-Str1/BAL#5 is not genetically supported (see discussion). Isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6, both phage susceptible, were obtained one month later (12 October 2017) and belonged to still another strain (Strain 3). Finally, on 8 February 2018, i.e., 16 days after the second phage therapy session (23 January 2018), an additional three isolates were obtained from BAL#9. Of these, isolate is9S-Str3/BAL#9 belonged to the same Strain 3 as isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6 and was phage susceptible as well, but isolates 7R and 8R belonged to still another strain (Strain 4) and were both resistant to the phage cocktail. Interestingly, the phage resistant Strain 4 isolates (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) contained mutations compared to the other six isolates that could explain the phage resistance observed for these two isolates (Figure 1): when Strain 1 was used as a reference, a C to A transition at position 1803 of the colicin I receptor gene caused a Tyr601->Stop transition. This receptor has been recognized as a phage receptor [7]. We further studied the course of infection by extracting DNA from the three available A. xylosoxidans-positive BAL samples (BAL#5, #6 and #9), and by quantifying DNA of the different strains by means of strain-specific qPCRs (Table 3 and Supplementary Methods) that had been developed on the basis of the obtained WGS data. qPCRs for Strain 2 and Strain 4 were only positive for the BALs from which these isolates had been cultured, respectively, BAL#5 and BAL#9. qPCR for Strain 1 was only positive for BAL#9, but not for BAL#5 from which isolate 3R had been cultured. qPCR for Strain 3 was positive for all three BALs, although no isolate belonging to Strain 3 was cultured from BAL#5. 3. Discussion and Conclusions We report the first case of decolonization of pandrug-resistant Achromobacter xylosoxidans after a long-term follow-up in a lung-transplanted patient receiving phage therapy. The first actual demonstration and usage of bacteriophages came almost simultaneously from two microbiologists in 1915 (Frederick Twort) and 1917 (Félix d’Hérelle) [8]. After extensive clinical use during the first half of the 20th century, their use has been progressively reduced, mainly due to the discovery, rapid spread, and ease of use of antibiotics for the treatment of infectious diseases [9]. However, bacteriophages gained a new surge of attention, due to the emergence and spread of pandrug-resistant bacteria [8]. Despite decades of clinical experience with bacteriophages in Eastern Europe, very few data are available regarding their clinical efficacy; besides, case-series and few randomized controlled trials (RCT) [10,11,12]. For instance, a RCT assessed the clinical outcome of patients receiving local application of a phage cocktail for the treatment of burn wound infection. This RCT is frequently cited to have shown no clinical benefits, as compared to standard of care. However, the phage titer that was applied (100 pfu/mL) [10], was so extremely low that it precludes to draw any conclusion from this study regarding the usefulness of phage therapy. The limited number of randomized clinical trials has been recently reviewed [13]. Phage therapy has also been used locally, e.g., for the treatment of bone and joint infections [14,15] or intravenously for the treatment of bloodstream infections [16,17]. Considering the high frequency of colonization and infection caused by antibiotic-resistant bacteria among cystic fibrosis and lung-transplanted patients, such as in our patient, the use of bacteriophages appears to be an appealing option in this population [18,19,20]. In the specific case of A. xylosoxidans, thus far a single case-report described the favorable outcome after the use of bacteriophages in a cystic fibrosis patient [21]. Our case-report highlights the many difficulties that need to be taken into account before considering the use of bacteriophages in a clinical setting. These include amongst others an important delay from clinical decision to regulatory agency authorization. Most problematic is the difficult interpretation of the clinical outcome after bacteriophage therapy, considering the long-term persisting airway colonization and the possible evolution of different A. xylosoxidans strains. The second therapeutic attempt relied on a massive lung instillation (injection of the bacteriophage solution in the patient’s lung during fibroscopy) followed by a two-week course of phage nebulization. We used a vibrating mesh nebulizer for phage delivery as this type of nebulizer has been shown to result in a lower phage titer reduction, as compared to jet nebulizers [22]. BAL#9, performed two weeks after the second round of phage therapy (8 February 2018) still grew 105 CFU/mL of A. xylosoxidans, represented by Strains 3 and 4 (according to culture) and by at least Strains 1, 3 and 4 (according to qPCR) (Table 1, Table 2 and Table 3). Sputum cultures remained positive for 18 months, but with only 103 CFU A. xylosoxidans/mL, suggesting a possible upper airway colonization, especially since no A. xylosoxidans could be isolated from BALs sampled in August 2019, and April 2020. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. To more precisely understand the discrepant outcome of our patient, another option would have been to search for active phages from the BAL samples throughout the study, but this was not performed. Despite the observed differences of a total of 367 SNPs between the four strains of eight A. xylosoxidans isolates from the patient, their overall similarity suggests that they originate from a single strain that colonized the patient (who was colonized with A. xylosoxidans years before the start of the phage therapy). Therefore, it is possible that some variants were present already before lung transplantation and before the start of phage therapy. Although it is not possible to exclude the simultaneous presence of these different strains on the basis of our culture results, because only one or two colonies were picked from each BAL culture, analysis by means of four strain-specific qPCR assays of the three BAL samples that were available for study, indicated that Strains 2 and 4 were probably prevalent at a particular moment of the course of infection, whereas Strains 1 and 3 could be identified during the full course of the infection. Phage resistant Strain 4 may have been selected during phage therapy. Phage training has been proposed to overcome phage resistance and increase infectivity against a given bacterial population [23]. However, training phages may take weeks, and therefore might be restricted to the treatment of chronic infections. Regarding more acute infections, the establishment of phage collections (such as the DSMZ, which provided bacteriophages for our patient) is of critical importance to provide phages active against a broad spectrum of bacterial isolates. An interesting point is the observation of two isolates from the same strain (is1S-Str1/BAL#3 and is3R-Str1/BAL#5) that differ regarding their phage susceptibility phenotype despite the absence of genetic support (identical genome). As observed with bacteria where emerging data point out that a resistance phenotype against a given antimicrobial may not be supported by a genotypic substratum [24,25], phage resistance without any change in genotype has also been documented [26]. These genotype/phenotype discrepancies are hypothesized to be possible through epigenetic regulation mechanisms, of which post-transcriptional regulation, which may influence phage receptor expression on the cell surface or phage receptor masking (through capsule synthesis). Further studies are required to better understand the dynamics of in vivo selection of phage-resistant bacteria. Because the patient’s medical condition improved anyhow, a few weeks after the second round of phage instillation, it is possible that phage therapy selected the phage-resistant A. xylosoxidans Strain 4, but that the mutation that caused phage resistance at the same time decreased the virulence of the strain as already described with other bacteria and phages [27,28,29]. Indeed, we observed several mutations which could explain the phage resistance of Strain 4, of which one was especially interesting, since it caused the gain of a stop codon in the colicin I receptor, which has been recognized as a phage receptor in other species [7]. Because in E. coli, the colicin I receptor plays a crucial role in iron transport across the outer membrane [7], loss of this function due to the observed mutation may have caused reduced fitness of the A. xylosoxidans mutant Strain 4. However, from a clinical point-of-view, one cannot exclude a spontaneous improvement, independently of phage therapy and despite months of fruitless antibiotic treatment, highlighting the dire need of randomized clinical trials in order to more convincingly assess the clinical efficacy of bacteriophages. In conclusion, we describe the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. Despite initial persisting airway colonization, the final clinical and microbiological outcome was favorable. Whole genome sequencing of eight A. xylosoxidans isolates and strain-specific qPCR experiments performed on BALs sampled before and after phage therapy allowed to more precisely study the dynamics of lung colonization by different isolates, susceptible or resistant to the applied phage cocktails. Acknowledgments The authors would like to thank L. Lécuyer and V. Boussaud (Unité de Transplantation, HEGP), B. Sabatier, V. Savoldelli, T. Caruba and P. Prognon (Pharmacie HEGP), P. Odou, C. Nassar, C. Berneron and M. Roche (Pharmacie Lille), and E. Deberranger (Hématologie Pédiatrique Lille), for their help in the management of the patient. Microbiological analysis was performed with a funding by the Association Robert Debré pour la Recherche Médicale. The authors would also like to thank Caroline Semaille and Nathalie Morgensztejn from the Agence Nationale de Sécurité du Médicament (ANSM) for their regulatory support. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Materials The following are available online at https://www.mdpi.com/1999-4915/13/1/60/s1, Figure S1: Antibiotic susceptibility testing (AST) of a pandrug-resistant Achromobacter xylosoxidans isolated during a bronchoalveolar lavage of a 12-year-old boy after double lung transplantation, Table S1: Variant calling for the eight Achromobacter xylosoxidans whole genome sequences, see excel file attached, named: “Supplementary Table S1. Variant calling.xls”. Click here for additional data file. Author Contributions D.L. (David Lebeaux) and M.V. conceptualized the study, validated the data, carried out formal analysis, prepared the initial draft, took care of project administration and supervised the study. Data were visualized by H.D. and Y.G. Software analysis was carried out by H.D., S.V., Y.G. D.L. (David Lebeaux), M.M., R.G., I.P., F.C., N.K., J.W., C.R., E.C., D.L. (Damien Lanoy), L.V.S., H.D., J.-P.P., N.D., S.V., Y.G., F.V.N., and M.V. contributed to basic methodology. Data curation was done by D.L. (David Lebeaux), M.M., C.T., S.V., Y.G., and M.V. Funding was acquired by D.L. (David Lebeaux), J.-L.M., F.V.N., and M.V. All authors took part in the investigation and in final reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement The study did not require ethical approval. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Phylogenetic tree built with Whole genome sequences of eight Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. The distance between samples reflects the number of different Single Nucleotide Polymorphisms (SNPs). Each A. xylosoxidans is named as follows: isolate number (is1 to is8) S (or R to depict susceptibility or resistance to phage cocktail)-Strain number/Number of BAL where it has been identified. viruses-13-00060-t001_Table 1Table 1 Results of bronchoalveolar lavages (BALs) and sputum analysis from May 2017, to April 2020, in 12-year-old lung transplanted patient who received two rounds of phage therapy for pandrug-resistant Achromobacter xylosoxidans colonization and infection. Date 19 May 2017 23 May 2017 6 June 2017 12 June 2017 25 July 2017 5 September 2017 8 September 2017 15 September 2017 12 October 2017 27 October 2017 23 January 2018 8 February 2018 5 March 2018 18 May 2018 19 February 2019 19 June 2019 27 August 2019 30 April 2020 Phage therapy ROUND 1 ROUND 2 Tigecycline from 29 June to 31 July Imipenem From 31 July 2017, to 16 February 2018 Number of days, relative to the first round of phage therapy −112 −108 −94 −88 −45 −3 +7 +37 +49 +137 +153 +178 +252 +529 +649 +718 +963 BAL BAL Number * #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 Leukocytes (/mm3) >1000 800 >1000 500 >1000 60 >1000 810 >1000 NA NA NA PMN cells (%) 90 88 90 91 43 30 80 69 82 NA NA NA Quantification of A. x culture (CFU/mL) 104 103 104 103 104 103 104 106 105 104 0 0 Other bacteria (CFU/mL) None None None None None None None None None None 104 S. a 106 S. a Sputum Leucocytes (/field) >25 >25 >25 >25 NA NA NA Quantification of A. x culture (CFU/mL) 105 105 105 106 103 103 103 Other bacteria (CFU/mL) None None None None 108 M. c None 106 S. a A. x: Achromobacter xylosoxidans; M. c: Moraxella catarrhalis; NA: non available; PMN: Polymorphonuclear; RBC: red blood cells; S. a: Staphylococcus aureus. : BAL number in bold indicates BALs for which qPCR had been carried out. viruses-13-00060-t002_Table 2Table 2 Eight Achromobacter xylosoxidans isolates for which whole genome sequencing was carried out. These were isolated from bronchoalveolar lavages in a 12-year-old boy after double lung transplantation. The patient received a first round of phage administration on 8 September 2017. A second round of phage treatment was performed on 23 January 2018. Complete Name Isolate Strain Source of Sampling Date of Sampling Whole Genome Sequence is1S-Str1/BAL#3 is1S Str1 BAL#3 25 July 2017 ERS5044236-UGAX1 is2S-Str2/BAL#5 is2S Str2 BAL#5 15 September 2017 ERS5044237-UGAX2 is3R-Str1/BAL#5 is3R Str1 BAL#5 15 September 2017 ERS5044238-UGAX3 is5S-Str3/BAL#6 is5S Str3 BAL#6 12 October 2017 ERS5044239-UGAX5 is6S-Str3/BAL#6 is6S Str3 BAL#6 12 October 2017 ERS5044240-UGAX6 is7R-Str4/BAL#9 is7R Str4 BAL#9 8 February 2018 ERS5044241-UGAX7 is8R-Str4/BAL#9 is8R Str4 BAL#9 8 February 2018 ERS5044242-UGAX8 is9S-Str3/BAL#9 is9S Str3 BAL#9 8 February 2018 ERS5044243-UGAX9 * Submitted at ENA, 1 September 2020. Study ID: PRJEB39103. BAL: Bronchoalveolar lavage, S: Phage susceptible, R: phage resistant. viruses-13-00060-t003_Table 3Table 3 Results of bacterial culture (grey-shading) and qPCR of three broncho-alveolar lavages for four different Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. Bronchoalveolar Lavage BAL#5 BAL#6 BAL#9 15 September 2017 12 October 2017 8 February 2018 qPCR for Strain Isolates Str1 is1S, is3R N NS P (34.7) Str2 is2S P (36.6) N N Str3 is5S, is6S, is9S P (32.7) P (28.9) P (27.7) Str4 is7R, is8R NS NS P (35.2) Grey-shaded box = isolate cultured from this BAL; N: negative (Cq-value > 40); NS: Nonspecific amplification (melting peak different from that of specific amplification); P: positive qPCR (Cq-value).	1.5 mg (milligrams).	DrugDosage	CC BY	33466377	19,957,163	2021-01-05
What was the dosage of drug 'VALACYCLOVIR HYDROCHLORIDE'?	A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient. Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains. 1. Case Presentation We report the case of a 12-year-old boy (35.5 kg), who received double lung transplantation in 25 March 2017, (at the age of 12) because of cystic fibrosis. Before transplantation, he was colonized with Aspergillus fumigatus, Achromobacter xylosoxidans (first colonization identified in 2013), and Pseudomonas aeruginosa. After transplantation, he experienced acute kidney injury requiring temporary haemodialysis, pulmonary embolism, and persisting airway colonization with A. fumigatus. He was discharged on 29 April 2017, with satisfactory respiratory status; he initially required no home oxygen therapy. His immunosuppressive regimen included prednisolone (25 mg/day), tacrolimus (1.5 mg bis in die (b.i.d.) with a target trough concentration of 6–8 ng/mL), mycophenolate mofetil (1 g b.i.d.) and his antimicrobial prophylaxis was cotrimoxazole (400/80 mg, three times a week), posaconazole (150 mg, oral suspension, ter in die (t.i.d.)), valacyclovir (500 mg b.i.d.), and intravenous immunoglobulins (15 g each three weeks). Between May and June 2017, he experienced progressive shortness of breath, cough and increased sputum production; he subsequently required the addition of 1–1.5 L/min oxygen therapy. Because of a stenosis of the surgical anastomosis of the main left bronchus, a dilatation during rigid bronchoscopy was performed on 19 May and a Montgomery stent was inserted on 2 June. Later on, a stenosis of the surgical anastomosis on the right bronchus intermedius required a dilatation during rigid bronchoscopy followed by the insertion of an Oki stent on 29 June. Several bronchoalveolar lavages (BAL), performed between May and June 2017, revealed inflammation (between 800 and >1000 leukocytes/mm3 with 88–90% of polymorphonuclear cells) and repeatedly grew pandrug-resistant A. xylosoxidans (Table 1 and Supplementary Figure S1) [1]. Lung biopsy performed on 19 June was consistent with lung infection: diffuse acute bronchiolitis with extension to peri-bronchiolar alveoli. Microbiological samples obtained from lung biopsies were negative (bacterial, mycological, and mycobacteriological cultures). The first antibiotic treatment consisted of intravenous tigecycline 50 mg b.i.d. from 29 June to 31 July. As BAL remained positive for A. xylosoxidans and the patient still required oxygen therapy; antibiotic treatment was subsequently switched to imipenem (1200 mg, t.i.d., ~100 mg/Kg/d) on 31 July 2017. Subsequent respiratory samples still grew A. xylosoxidans and the respiratory status did not improve (requiring oxygen at home, 1 L/min). Furthermore, despite antibiotic treatment and correct drainage of the right superior lobe, lung consolidation and micronodules remained. Considering the failure of antibiotic treatment, phage therapy was proposed to the patient and his family, after multidisciplinary discussion. A first cocktail (APC 1.1) containing three lytic phages (JWDelta, JWT and 2-1) active against A. xylosoxidans isolate is1S (designated such as later), selected from the DSMZ collection (Braunschweig, Germany) [2,3], was prepared at the Laboratory for Bacteriology Research (Ghent University, Belgium) and the Laboratory for Molecular and Cellular Technology (Queen Astrid Military Hospital, Brussels, Belgium) using the above-mentioned bacterial isolate, as previously described [4]. The production was completed on 23 August with a final phage titer of 4 × 1010 plaque forming units (pfu)/mL defined by double-agar overlay method [4], an endotoxin level of 4000 EU/mL (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, GenScript, Piscataway Township, NJ, USA) and a pH of 7.3 (in sterile PBS). Regulatory permission for phage importation was obtained on 1 September 2017, from the Agence Nationale de Sécurité du Médicament (ANSM). A first round of phage administration was performed, consisting of 3 nebulizations/day of 5 mL of the filter-sterilized (Sartorius 0.2 µm) phage solution, tenfold diluted in sterile saline on 8 and 9 September, using a vibrating mesh nebulizer (eFLOW rapid, PARI, PARI GmbH, Germany) with a mouth piece. Immediate tolerance was perfect, but no clinical improvement was noted and culture of subsequent respiratory samples remained positive for A. xylosoxidans (104 CFU/mL in the BAL of 15 September). A second cocktail (APC 2.1) was produced, in which phage JWalpha was added to the three lytic phages of cocktail APC 1.1, to improve the therapeutic efficacy. The production was completed on 22 December with a final phage titer of 5 × 109 pfu/mL, an endotoxin level of 1760 EU/mL and a pH of 6.95 (in sterile PBS). Regulatory permission was obtained for phage importation on 16 January 2018 (ANSM). On 23 January, during therapeutic bronchoscopy under general anesthesia, 30 mL of APC2.1, tenfold diluted (in sterile saline) and filter-sterilized, was instilled in each pulmonary lobe through the fibroscope. Immediate tolerance was perfect and the patient was discharged on 24 January with continued phage nebulization at home: three times a day 5 mL of preparation A using a vibrating mesh nebulizer (eFLOW rapid, PARI) with a mouth piece for 14 days until 6 February. Clinical tolerance was perfect again, but the initial clinical status remained unchanged. BAL performed on 8 February 2018, two days after stopping the phage treatment, still grew 105 cfu/mL of A. xylosoxidans. Subsequently, the patient’s respiratory condition slowly improved and oxygen therapy was stopped on 15 February 2018. No clinical worsening was observed after imipenem interruption on 16 February 2018. Sputum culture remained positive for A. xylosoxidans but with a low bacterial density (103 CFU/mL in March and May 2018, and June 2019) and no A. xylosoxidans could be isolated from BALs#10 and #11, sampled on August 2019, and April 2020, respectively (Table 1). The last pulmonary function tests, performed on 17 October 2019, showed best results since lung transplantation, with an FEV1 of 79% and a forced vital capacity of 85%. 2. Microbiological Analysis of Eight A. xylosoxidans Isolates As we observed discrepant outcome in our patient (clinical improvement on the one hand but persisting low-grade airway colonization on the other hand), we decided to study the eight available A. xylosoxidans isolates (is1 to is9) from the patient’s colonization obtained by random picking from the culture plates from 4 distinct BALs (Table 2). These eight isolates, taken before, during and after the phage therapy period (Table 2), could not be differentiated from each other by means of MALDI-TOF [5] or McRAPD typing [6] and all belonged to the epidemic clone, already described previously among Belgian CF patients [5]. Whole genome sequencing (WGS) and de novo genome assembly for these eight isolates was carried out (Supplementary Methods and Table S1). The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Total genome length for all eight isolates was 6.44–6.50 Mbp, with a GC content of 67.5%. All eight isolates showed most similarity with reference genome GCF_008432465 (AX1), which corresponds to the type strain of A. xylosoxidans subsp. xylosoxidans (ATCC 27061T, CCUG 12689T, DSMZ 10346T, LMG 1863T, and NCTC 10807T), i.e., between 88.38 and 89.60% on the basis of mapped sequence reads, and between 98.71% and 98.76% on the basis of average nucleotide identity (ANI). Indeed, the overall similarity between the genome sequences of the eight isolates was high, i.e., 99.60%, as assessed by digital DNA hybridization, and more than 99.97% according to ANI-analysis. This was confirmed by variant calling where we observed that the isolates differed from each other only for a total of 367 SNPs, whereas a total of 59,452 SNPs were observed between the isolates and the best matching reference genome (AX1) (Figure 1). The susceptibility phenotype of each of the eight isolates was assessed by spot-test against the phage cocktail APC 2.1 and expressed as “S” when susceptible or “R” when resistant (e.g., “is2S” means that isolate number 2 was susceptible to the phage cocktail) (Table 2 and Supplementary Methods). Among these eight isolates, analysis based on variant calling furthermore revealed four closely related but clearly delineated strains, i.e., clones that differ genetically from other clones, with different phage susceptibility phenotype (Table 2 and Figure 1): Strain 1 (Str1), contained isolates 1 and 3 (is1S and is3R), found in BAL#3 (is1S-Str1/BAL#3) and BAL#5 (is3R-Str1/BAL#5), respectively. Strain 2 (Str2) contained isolate 2 (is2S) from BAL#5 (is2S-Str2/BAL#5). Strain 3 (Str3) contained isolates 5, 6, and 9 (is5S, is6S and is9S), which were found in BAL#6 (is5S-Str3/BAL#6, is6S-Str3/BAL#6) and BAL#9 (is9S-Str3/BAL#9). Strain 4 contained isolates 7 and 8 (is7R and is8R), found in BAL#9 (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) (Table 2). Isolate is1S-Str1/BAL#3 was obtained on 25 July 2017, i.e., 45 days before the first phage therapy session (8 September 2017) and was 100% identical to is3R-Str1/BAL#5 isolated seven days (15 September 2017) after the first phage therapy session (Figure 1). Since both isolates is1S-Str1/BAL#3 and is3R-Str1/BAL#5 are 100% identical, this means that the phage resistance mechanism of isolate is3R-Str1/BAL#5 is not genetically supported (see discussion). Isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6, both phage susceptible, were obtained one month later (12 October 2017) and belonged to still another strain (Strain 3). Finally, on 8 February 2018, i.e., 16 days after the second phage therapy session (23 January 2018), an additional three isolates were obtained from BAL#9. Of these, isolate is9S-Str3/BAL#9 belonged to the same Strain 3 as isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6 and was phage susceptible as well, but isolates 7R and 8R belonged to still another strain (Strain 4) and were both resistant to the phage cocktail. Interestingly, the phage resistant Strain 4 isolates (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) contained mutations compared to the other six isolates that could explain the phage resistance observed for these two isolates (Figure 1): when Strain 1 was used as a reference, a C to A transition at position 1803 of the colicin I receptor gene caused a Tyr601->Stop transition. This receptor has been recognized as a phage receptor [7]. We further studied the course of infection by extracting DNA from the three available A. xylosoxidans-positive BAL samples (BAL#5, #6 and #9), and by quantifying DNA of the different strains by means of strain-specific qPCRs (Table 3 and Supplementary Methods) that had been developed on the basis of the obtained WGS data. qPCRs for Strain 2 and Strain 4 were only positive for the BALs from which these isolates had been cultured, respectively, BAL#5 and BAL#9. qPCR for Strain 1 was only positive for BAL#9, but not for BAL#5 from which isolate 3R had been cultured. qPCR for Strain 3 was positive for all three BALs, although no isolate belonging to Strain 3 was cultured from BAL#5. 3. Discussion and Conclusions We report the first case of decolonization of pandrug-resistant Achromobacter xylosoxidans after a long-term follow-up in a lung-transplanted patient receiving phage therapy. The first actual demonstration and usage of bacteriophages came almost simultaneously from two microbiologists in 1915 (Frederick Twort) and 1917 (Félix d’Hérelle) [8]. After extensive clinical use during the first half of the 20th century, their use has been progressively reduced, mainly due to the discovery, rapid spread, and ease of use of antibiotics for the treatment of infectious diseases [9]. However, bacteriophages gained a new surge of attention, due to the emergence and spread of pandrug-resistant bacteria [8]. Despite decades of clinical experience with bacteriophages in Eastern Europe, very few data are available regarding their clinical efficacy; besides, case-series and few randomized controlled trials (RCT) [10,11,12]. For instance, a RCT assessed the clinical outcome of patients receiving local application of a phage cocktail for the treatment of burn wound infection. This RCT is frequently cited to have shown no clinical benefits, as compared to standard of care. However, the phage titer that was applied (100 pfu/mL) [10], was so extremely low that it precludes to draw any conclusion from this study regarding the usefulness of phage therapy. The limited number of randomized clinical trials has been recently reviewed [13]. Phage therapy has also been used locally, e.g., for the treatment of bone and joint infections [14,15] or intravenously for the treatment of bloodstream infections [16,17]. Considering the high frequency of colonization and infection caused by antibiotic-resistant bacteria among cystic fibrosis and lung-transplanted patients, such as in our patient, the use of bacteriophages appears to be an appealing option in this population [18,19,20]. In the specific case of A. xylosoxidans, thus far a single case-report described the favorable outcome after the use of bacteriophages in a cystic fibrosis patient [21]. Our case-report highlights the many difficulties that need to be taken into account before considering the use of bacteriophages in a clinical setting. These include amongst others an important delay from clinical decision to regulatory agency authorization. Most problematic is the difficult interpretation of the clinical outcome after bacteriophage therapy, considering the long-term persisting airway colonization and the possible evolution of different A. xylosoxidans strains. The second therapeutic attempt relied on a massive lung instillation (injection of the bacteriophage solution in the patient’s lung during fibroscopy) followed by a two-week course of phage nebulization. We used a vibrating mesh nebulizer for phage delivery as this type of nebulizer has been shown to result in a lower phage titer reduction, as compared to jet nebulizers [22]. BAL#9, performed two weeks after the second round of phage therapy (8 February 2018) still grew 105 CFU/mL of A. xylosoxidans, represented by Strains 3 and 4 (according to culture) and by at least Strains 1, 3 and 4 (according to qPCR) (Table 1, Table 2 and Table 3). Sputum cultures remained positive for 18 months, but with only 103 CFU A. xylosoxidans/mL, suggesting a possible upper airway colonization, especially since no A. xylosoxidans could be isolated from BALs sampled in August 2019, and April 2020. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. To more precisely understand the discrepant outcome of our patient, another option would have been to search for active phages from the BAL samples throughout the study, but this was not performed. Despite the observed differences of a total of 367 SNPs between the four strains of eight A. xylosoxidans isolates from the patient, their overall similarity suggests that they originate from a single strain that colonized the patient (who was colonized with A. xylosoxidans years before the start of the phage therapy). Therefore, it is possible that some variants were present already before lung transplantation and before the start of phage therapy. Although it is not possible to exclude the simultaneous presence of these different strains on the basis of our culture results, because only one or two colonies were picked from each BAL culture, analysis by means of four strain-specific qPCR assays of the three BAL samples that were available for study, indicated that Strains 2 and 4 were probably prevalent at a particular moment of the course of infection, whereas Strains 1 and 3 could be identified during the full course of the infection. Phage resistant Strain 4 may have been selected during phage therapy. Phage training has been proposed to overcome phage resistance and increase infectivity against a given bacterial population [23]. However, training phages may take weeks, and therefore might be restricted to the treatment of chronic infections. Regarding more acute infections, the establishment of phage collections (such as the DSMZ, which provided bacteriophages for our patient) is of critical importance to provide phages active against a broad spectrum of bacterial isolates. An interesting point is the observation of two isolates from the same strain (is1S-Str1/BAL#3 and is3R-Str1/BAL#5) that differ regarding their phage susceptibility phenotype despite the absence of genetic support (identical genome). As observed with bacteria where emerging data point out that a resistance phenotype against a given antimicrobial may not be supported by a genotypic substratum [24,25], phage resistance without any change in genotype has also been documented [26]. These genotype/phenotype discrepancies are hypothesized to be possible through epigenetic regulation mechanisms, of which post-transcriptional regulation, which may influence phage receptor expression on the cell surface or phage receptor masking (through capsule synthesis). Further studies are required to better understand the dynamics of in vivo selection of phage-resistant bacteria. Because the patient’s medical condition improved anyhow, a few weeks after the second round of phage instillation, it is possible that phage therapy selected the phage-resistant A. xylosoxidans Strain 4, but that the mutation that caused phage resistance at the same time decreased the virulence of the strain as already described with other bacteria and phages [27,28,29]. Indeed, we observed several mutations which could explain the phage resistance of Strain 4, of which one was especially interesting, since it caused the gain of a stop codon in the colicin I receptor, which has been recognized as a phage receptor in other species [7]. Because in E. coli, the colicin I receptor plays a crucial role in iron transport across the outer membrane [7], loss of this function due to the observed mutation may have caused reduced fitness of the A. xylosoxidans mutant Strain 4. However, from a clinical point-of-view, one cannot exclude a spontaneous improvement, independently of phage therapy and despite months of fruitless antibiotic treatment, highlighting the dire need of randomized clinical trials in order to more convincingly assess the clinical efficacy of bacteriophages. In conclusion, we describe the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. Despite initial persisting airway colonization, the final clinical and microbiological outcome was favorable. Whole genome sequencing of eight A. xylosoxidans isolates and strain-specific qPCR experiments performed on BALs sampled before and after phage therapy allowed to more precisely study the dynamics of lung colonization by different isolates, susceptible or resistant to the applied phage cocktails. Acknowledgments The authors would like to thank L. Lécuyer and V. Boussaud (Unité de Transplantation, HEGP), B. Sabatier, V. Savoldelli, T. Caruba and P. Prognon (Pharmacie HEGP), P. Odou, C. Nassar, C. Berneron and M. Roche (Pharmacie Lille), and E. Deberranger (Hématologie Pédiatrique Lille), for their help in the management of the patient. Microbiological analysis was performed with a funding by the Association Robert Debré pour la Recherche Médicale. The authors would also like to thank Caroline Semaille and Nathalie Morgensztejn from the Agence Nationale de Sécurité du Médicament (ANSM) for their regulatory support. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Materials The following are available online at https://www.mdpi.com/1999-4915/13/1/60/s1, Figure S1: Antibiotic susceptibility testing (AST) of a pandrug-resistant Achromobacter xylosoxidans isolated during a bronchoalveolar lavage of a 12-year-old boy after double lung transplantation, Table S1: Variant calling for the eight Achromobacter xylosoxidans whole genome sequences, see excel file attached, named: “Supplementary Table S1. Variant calling.xls”. Click here for additional data file. Author Contributions D.L. (David Lebeaux) and M.V. conceptualized the study, validated the data, carried out formal analysis, prepared the initial draft, took care of project administration and supervised the study. Data were visualized by H.D. and Y.G. Software analysis was carried out by H.D., S.V., Y.G. D.L. (David Lebeaux), M.M., R.G., I.P., F.C., N.K., J.W., C.R., E.C., D.L. (Damien Lanoy), L.V.S., H.D., J.-P.P., N.D., S.V., Y.G., F.V.N., and M.V. contributed to basic methodology. Data curation was done by D.L. (David Lebeaux), M.M., C.T., S.V., Y.G., and M.V. Funding was acquired by D.L. (David Lebeaux), J.-L.M., F.V.N., and M.V. All authors took part in the investigation and in final reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement The study did not require ethical approval. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Phylogenetic tree built with Whole genome sequences of eight Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. The distance between samples reflects the number of different Single Nucleotide Polymorphisms (SNPs). Each A. xylosoxidans is named as follows: isolate number (is1 to is8) S (or R to depict susceptibility or resistance to phage cocktail)-Strain number/Number of BAL where it has been identified. viruses-13-00060-t001_Table 1Table 1 Results of bronchoalveolar lavages (BALs) and sputum analysis from May 2017, to April 2020, in 12-year-old lung transplanted patient who received two rounds of phage therapy for pandrug-resistant Achromobacter xylosoxidans colonization and infection. Date 19 May 2017 23 May 2017 6 June 2017 12 June 2017 25 July 2017 5 September 2017 8 September 2017 15 September 2017 12 October 2017 27 October 2017 23 January 2018 8 February 2018 5 March 2018 18 May 2018 19 February 2019 19 June 2019 27 August 2019 30 April 2020 Phage therapy ROUND 1 ROUND 2 Tigecycline from 29 June to 31 July Imipenem From 31 July 2017, to 16 February 2018 Number of days, relative to the first round of phage therapy −112 −108 −94 −88 −45 −3 +7 +37 +49 +137 +153 +178 +252 +529 +649 +718 +963 BAL BAL Number * #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 Leukocytes (/mm3) >1000 800 >1000 500 >1000 60 >1000 810 >1000 NA NA NA PMN cells (%) 90 88 90 91 43 30 80 69 82 NA NA NA Quantification of A. x culture (CFU/mL) 104 103 104 103 104 103 104 106 105 104 0 0 Other bacteria (CFU/mL) None None None None None None None None None None 104 S. a 106 S. a Sputum Leucocytes (/field) >25 >25 >25 >25 NA NA NA Quantification of A. x culture (CFU/mL) 105 105 105 106 103 103 103 Other bacteria (CFU/mL) None None None None 108 M. c None 106 S. a A. x: Achromobacter xylosoxidans; M. c: Moraxella catarrhalis; NA: non available; PMN: Polymorphonuclear; RBC: red blood cells; S. a: Staphylococcus aureus. : BAL number in bold indicates BALs for which qPCR had been carried out. viruses-13-00060-t002_Table 2Table 2 Eight Achromobacter xylosoxidans isolates for which whole genome sequencing was carried out. These were isolated from bronchoalveolar lavages in a 12-year-old boy after double lung transplantation. The patient received a first round of phage administration on 8 September 2017. A second round of phage treatment was performed on 23 January 2018. Complete Name Isolate Strain Source of Sampling Date of Sampling Whole Genome Sequence is1S-Str1/BAL#3 is1S Str1 BAL#3 25 July 2017 ERS5044236-UGAX1 is2S-Str2/BAL#5 is2S Str2 BAL#5 15 September 2017 ERS5044237-UGAX2 is3R-Str1/BAL#5 is3R Str1 BAL#5 15 September 2017 ERS5044238-UGAX3 is5S-Str3/BAL#6 is5S Str3 BAL#6 12 October 2017 ERS5044239-UGAX5 is6S-Str3/BAL#6 is6S Str3 BAL#6 12 October 2017 ERS5044240-UGAX6 is7R-Str4/BAL#9 is7R Str4 BAL#9 8 February 2018 ERS5044241-UGAX7 is8R-Str4/BAL#9 is8R Str4 BAL#9 8 February 2018 ERS5044242-UGAX8 is9S-Str3/BAL#9 is9S Str3 BAL#9 8 February 2018 ERS5044243-UGAX9 * Submitted at ENA, 1 September 2020. Study ID: PRJEB39103. BAL: Bronchoalveolar lavage, S: Phage susceptible, R: phage resistant. viruses-13-00060-t003_Table 3Table 3 Results of bacterial culture (grey-shading) and qPCR of three broncho-alveolar lavages for four different Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. Bronchoalveolar Lavage BAL#5 BAL#6 BAL#9 15 September 2017 12 October 2017 8 February 2018 qPCR for Strain Isolates Str1 is1S, is3R N NS P (34.7) Str2 is2S P (36.6) N N Str3 is5S, is6S, is9S P (32.7) P (28.9) P (27.7) Str4 is7R, is8R NS NS P (35.2) Grey-shaded box = isolate cultured from this BAL; N: negative (Cq-value > 40); NS: Nonspecific amplification (melting peak different from that of specific amplification); P: positive qPCR (Cq-value).	500 mg (milligrams).	DrugDosage	CC BY	33466377	19,957,163	2021-01-05
What was the outcome of reaction 'Achromobacter infection'?	A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient. Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains. 1. Case Presentation We report the case of a 12-year-old boy (35.5 kg), who received double lung transplantation in 25 March 2017, (at the age of 12) because of cystic fibrosis. Before transplantation, he was colonized with Aspergillus fumigatus, Achromobacter xylosoxidans (first colonization identified in 2013), and Pseudomonas aeruginosa. After transplantation, he experienced acute kidney injury requiring temporary haemodialysis, pulmonary embolism, and persisting airway colonization with A. fumigatus. He was discharged on 29 April 2017, with satisfactory respiratory status; he initially required no home oxygen therapy. His immunosuppressive regimen included prednisolone (25 mg/day), tacrolimus (1.5 mg bis in die (b.i.d.) with a target trough concentration of 6–8 ng/mL), mycophenolate mofetil (1 g b.i.d.) and his antimicrobial prophylaxis was cotrimoxazole (400/80 mg, three times a week), posaconazole (150 mg, oral suspension, ter in die (t.i.d.)), valacyclovir (500 mg b.i.d.), and intravenous immunoglobulins (15 g each three weeks). Between May and June 2017, he experienced progressive shortness of breath, cough and increased sputum production; he subsequently required the addition of 1–1.5 L/min oxygen therapy. Because of a stenosis of the surgical anastomosis of the main left bronchus, a dilatation during rigid bronchoscopy was performed on 19 May and a Montgomery stent was inserted on 2 June. Later on, a stenosis of the surgical anastomosis on the right bronchus intermedius required a dilatation during rigid bronchoscopy followed by the insertion of an Oki stent on 29 June. Several bronchoalveolar lavages (BAL), performed between May and June 2017, revealed inflammation (between 800 and >1000 leukocytes/mm3 with 88–90% of polymorphonuclear cells) and repeatedly grew pandrug-resistant A. xylosoxidans (Table 1 and Supplementary Figure S1) [1]. Lung biopsy performed on 19 June was consistent with lung infection: diffuse acute bronchiolitis with extension to peri-bronchiolar alveoli. Microbiological samples obtained from lung biopsies were negative (bacterial, mycological, and mycobacteriological cultures). The first antibiotic treatment consisted of intravenous tigecycline 50 mg b.i.d. from 29 June to 31 July. As BAL remained positive for A. xylosoxidans and the patient still required oxygen therapy; antibiotic treatment was subsequently switched to imipenem (1200 mg, t.i.d., ~100 mg/Kg/d) on 31 July 2017. Subsequent respiratory samples still grew A. xylosoxidans and the respiratory status did not improve (requiring oxygen at home, 1 L/min). Furthermore, despite antibiotic treatment and correct drainage of the right superior lobe, lung consolidation and micronodules remained. Considering the failure of antibiotic treatment, phage therapy was proposed to the patient and his family, after multidisciplinary discussion. A first cocktail (APC 1.1) containing three lytic phages (JWDelta, JWT and 2-1) active against A. xylosoxidans isolate is1S (designated such as later), selected from the DSMZ collection (Braunschweig, Germany) [2,3], was prepared at the Laboratory for Bacteriology Research (Ghent University, Belgium) and the Laboratory for Molecular and Cellular Technology (Queen Astrid Military Hospital, Brussels, Belgium) using the above-mentioned bacterial isolate, as previously described [4]. The production was completed on 23 August with a final phage titer of 4 × 1010 plaque forming units (pfu)/mL defined by double-agar overlay method [4], an endotoxin level of 4000 EU/mL (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, GenScript, Piscataway Township, NJ, USA) and a pH of 7.3 (in sterile PBS). Regulatory permission for phage importation was obtained on 1 September 2017, from the Agence Nationale de Sécurité du Médicament (ANSM). A first round of phage administration was performed, consisting of 3 nebulizations/day of 5 mL of the filter-sterilized (Sartorius 0.2 µm) phage solution, tenfold diluted in sterile saline on 8 and 9 September, using a vibrating mesh nebulizer (eFLOW rapid, PARI, PARI GmbH, Germany) with a mouth piece. Immediate tolerance was perfect, but no clinical improvement was noted and culture of subsequent respiratory samples remained positive for A. xylosoxidans (104 CFU/mL in the BAL of 15 September). A second cocktail (APC 2.1) was produced, in which phage JWalpha was added to the three lytic phages of cocktail APC 1.1, to improve the therapeutic efficacy. The production was completed on 22 December with a final phage titer of 5 × 109 pfu/mL, an endotoxin level of 1760 EU/mL and a pH of 6.95 (in sterile PBS). Regulatory permission was obtained for phage importation on 16 January 2018 (ANSM). On 23 January, during therapeutic bronchoscopy under general anesthesia, 30 mL of APC2.1, tenfold diluted (in sterile saline) and filter-sterilized, was instilled in each pulmonary lobe through the fibroscope. Immediate tolerance was perfect and the patient was discharged on 24 January with continued phage nebulization at home: three times a day 5 mL of preparation A using a vibrating mesh nebulizer (eFLOW rapid, PARI) with a mouth piece for 14 days until 6 February. Clinical tolerance was perfect again, but the initial clinical status remained unchanged. BAL performed on 8 February 2018, two days after stopping the phage treatment, still grew 105 cfu/mL of A. xylosoxidans. Subsequently, the patient’s respiratory condition slowly improved and oxygen therapy was stopped on 15 February 2018. No clinical worsening was observed after imipenem interruption on 16 February 2018. Sputum culture remained positive for A. xylosoxidans but with a low bacterial density (103 CFU/mL in March and May 2018, and June 2019) and no A. xylosoxidans could be isolated from BALs#10 and #11, sampled on August 2019, and April 2020, respectively (Table 1). The last pulmonary function tests, performed on 17 October 2019, showed best results since lung transplantation, with an FEV1 of 79% and a forced vital capacity of 85%. 2. Microbiological Analysis of Eight A. xylosoxidans Isolates As we observed discrepant outcome in our patient (clinical improvement on the one hand but persisting low-grade airway colonization on the other hand), we decided to study the eight available A. xylosoxidans isolates (is1 to is9) from the patient’s colonization obtained by random picking from the culture plates from 4 distinct BALs (Table 2). These eight isolates, taken before, during and after the phage therapy period (Table 2), could not be differentiated from each other by means of MALDI-TOF [5] or McRAPD typing [6] and all belonged to the epidemic clone, already described previously among Belgian CF patients [5]. Whole genome sequencing (WGS) and de novo genome assembly for these eight isolates was carried out (Supplementary Methods and Table S1). The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Total genome length for all eight isolates was 6.44–6.50 Mbp, with a GC content of 67.5%. All eight isolates showed most similarity with reference genome GCF_008432465 (AX1), which corresponds to the type strain of A. xylosoxidans subsp. xylosoxidans (ATCC 27061T, CCUG 12689T, DSMZ 10346T, LMG 1863T, and NCTC 10807T), i.e., between 88.38 and 89.60% on the basis of mapped sequence reads, and between 98.71% and 98.76% on the basis of average nucleotide identity (ANI). Indeed, the overall similarity between the genome sequences of the eight isolates was high, i.e., 99.60%, as assessed by digital DNA hybridization, and more than 99.97% according to ANI-analysis. This was confirmed by variant calling where we observed that the isolates differed from each other only for a total of 367 SNPs, whereas a total of 59,452 SNPs were observed between the isolates and the best matching reference genome (AX1) (Figure 1). The susceptibility phenotype of each of the eight isolates was assessed by spot-test against the phage cocktail APC 2.1 and expressed as “S” when susceptible or “R” when resistant (e.g., “is2S” means that isolate number 2 was susceptible to the phage cocktail) (Table 2 and Supplementary Methods). Among these eight isolates, analysis based on variant calling furthermore revealed four closely related but clearly delineated strains, i.e., clones that differ genetically from other clones, with different phage susceptibility phenotype (Table 2 and Figure 1): Strain 1 (Str1), contained isolates 1 and 3 (is1S and is3R), found in BAL#3 (is1S-Str1/BAL#3) and BAL#5 (is3R-Str1/BAL#5), respectively. Strain 2 (Str2) contained isolate 2 (is2S) from BAL#5 (is2S-Str2/BAL#5). Strain 3 (Str3) contained isolates 5, 6, and 9 (is5S, is6S and is9S), which were found in BAL#6 (is5S-Str3/BAL#6, is6S-Str3/BAL#6) and BAL#9 (is9S-Str3/BAL#9). Strain 4 contained isolates 7 and 8 (is7R and is8R), found in BAL#9 (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) (Table 2). Isolate is1S-Str1/BAL#3 was obtained on 25 July 2017, i.e., 45 days before the first phage therapy session (8 September 2017) and was 100% identical to is3R-Str1/BAL#5 isolated seven days (15 September 2017) after the first phage therapy session (Figure 1). Since both isolates is1S-Str1/BAL#3 and is3R-Str1/BAL#5 are 100% identical, this means that the phage resistance mechanism of isolate is3R-Str1/BAL#5 is not genetically supported (see discussion). Isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6, both phage susceptible, were obtained one month later (12 October 2017) and belonged to still another strain (Strain 3). Finally, on 8 February 2018, i.e., 16 days after the second phage therapy session (23 January 2018), an additional three isolates were obtained from BAL#9. Of these, isolate is9S-Str3/BAL#9 belonged to the same Strain 3 as isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6 and was phage susceptible as well, but isolates 7R and 8R belonged to still another strain (Strain 4) and were both resistant to the phage cocktail. Interestingly, the phage resistant Strain 4 isolates (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) contained mutations compared to the other six isolates that could explain the phage resistance observed for these two isolates (Figure 1): when Strain 1 was used as a reference, a C to A transition at position 1803 of the colicin I receptor gene caused a Tyr601->Stop transition. This receptor has been recognized as a phage receptor [7]. We further studied the course of infection by extracting DNA from the three available A. xylosoxidans-positive BAL samples (BAL#5, #6 and #9), and by quantifying DNA of the different strains by means of strain-specific qPCRs (Table 3 and Supplementary Methods) that had been developed on the basis of the obtained WGS data. qPCRs for Strain 2 and Strain 4 were only positive for the BALs from which these isolates had been cultured, respectively, BAL#5 and BAL#9. qPCR for Strain 1 was only positive for BAL#9, but not for BAL#5 from which isolate 3R had been cultured. qPCR for Strain 3 was positive for all three BALs, although no isolate belonging to Strain 3 was cultured from BAL#5. 3. Discussion and Conclusions We report the first case of decolonization of pandrug-resistant Achromobacter xylosoxidans after a long-term follow-up in a lung-transplanted patient receiving phage therapy. The first actual demonstration and usage of bacteriophages came almost simultaneously from two microbiologists in 1915 (Frederick Twort) and 1917 (Félix d’Hérelle) [8]. After extensive clinical use during the first half of the 20th century, their use has been progressively reduced, mainly due to the discovery, rapid spread, and ease of use of antibiotics for the treatment of infectious diseases [9]. However, bacteriophages gained a new surge of attention, due to the emergence and spread of pandrug-resistant bacteria [8]. Despite decades of clinical experience with bacteriophages in Eastern Europe, very few data are available regarding their clinical efficacy; besides, case-series and few randomized controlled trials (RCT) [10,11,12]. For instance, a RCT assessed the clinical outcome of patients receiving local application of a phage cocktail for the treatment of burn wound infection. This RCT is frequently cited to have shown no clinical benefits, as compared to standard of care. However, the phage titer that was applied (100 pfu/mL) [10], was so extremely low that it precludes to draw any conclusion from this study regarding the usefulness of phage therapy. The limited number of randomized clinical trials has been recently reviewed [13]. Phage therapy has also been used locally, e.g., for the treatment of bone and joint infections [14,15] or intravenously for the treatment of bloodstream infections [16,17]. Considering the high frequency of colonization and infection caused by antibiotic-resistant bacteria among cystic fibrosis and lung-transplanted patients, such as in our patient, the use of bacteriophages appears to be an appealing option in this population [18,19,20]. In the specific case of A. xylosoxidans, thus far a single case-report described the favorable outcome after the use of bacteriophages in a cystic fibrosis patient [21]. Our case-report highlights the many difficulties that need to be taken into account before considering the use of bacteriophages in a clinical setting. These include amongst others an important delay from clinical decision to regulatory agency authorization. Most problematic is the difficult interpretation of the clinical outcome after bacteriophage therapy, considering the long-term persisting airway colonization and the possible evolution of different A. xylosoxidans strains. The second therapeutic attempt relied on a massive lung instillation (injection of the bacteriophage solution in the patient’s lung during fibroscopy) followed by a two-week course of phage nebulization. We used a vibrating mesh nebulizer for phage delivery as this type of nebulizer has been shown to result in a lower phage titer reduction, as compared to jet nebulizers [22]. BAL#9, performed two weeks after the second round of phage therapy (8 February 2018) still grew 105 CFU/mL of A. xylosoxidans, represented by Strains 3 and 4 (according to culture) and by at least Strains 1, 3 and 4 (according to qPCR) (Table 1, Table 2 and Table 3). Sputum cultures remained positive for 18 months, but with only 103 CFU A. xylosoxidans/mL, suggesting a possible upper airway colonization, especially since no A. xylosoxidans could be isolated from BALs sampled in August 2019, and April 2020. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. To more precisely understand the discrepant outcome of our patient, another option would have been to search for active phages from the BAL samples throughout the study, but this was not performed. Despite the observed differences of a total of 367 SNPs between the four strains of eight A. xylosoxidans isolates from the patient, their overall similarity suggests that they originate from a single strain that colonized the patient (who was colonized with A. xylosoxidans years before the start of the phage therapy). Therefore, it is possible that some variants were present already before lung transplantation and before the start of phage therapy. Although it is not possible to exclude the simultaneous presence of these different strains on the basis of our culture results, because only one or two colonies were picked from each BAL culture, analysis by means of four strain-specific qPCR assays of the three BAL samples that were available for study, indicated that Strains 2 and 4 were probably prevalent at a particular moment of the course of infection, whereas Strains 1 and 3 could be identified during the full course of the infection. Phage resistant Strain 4 may have been selected during phage therapy. Phage training has been proposed to overcome phage resistance and increase infectivity against a given bacterial population [23]. However, training phages may take weeks, and therefore might be restricted to the treatment of chronic infections. Regarding more acute infections, the establishment of phage collections (such as the DSMZ, which provided bacteriophages for our patient) is of critical importance to provide phages active against a broad spectrum of bacterial isolates. An interesting point is the observation of two isolates from the same strain (is1S-Str1/BAL#3 and is3R-Str1/BAL#5) that differ regarding their phage susceptibility phenotype despite the absence of genetic support (identical genome). As observed with bacteria where emerging data point out that a resistance phenotype against a given antimicrobial may not be supported by a genotypic substratum [24,25], phage resistance without any change in genotype has also been documented [26]. These genotype/phenotype discrepancies are hypothesized to be possible through epigenetic regulation mechanisms, of which post-transcriptional regulation, which may influence phage receptor expression on the cell surface or phage receptor masking (through capsule synthesis). Further studies are required to better understand the dynamics of in vivo selection of phage-resistant bacteria. Because the patient’s medical condition improved anyhow, a few weeks after the second round of phage instillation, it is possible that phage therapy selected the phage-resistant A. xylosoxidans Strain 4, but that the mutation that caused phage resistance at the same time decreased the virulence of the strain as already described with other bacteria and phages [27,28,29]. Indeed, we observed several mutations which could explain the phage resistance of Strain 4, of which one was especially interesting, since it caused the gain of a stop codon in the colicin I receptor, which has been recognized as a phage receptor in other species [7]. Because in E. coli, the colicin I receptor plays a crucial role in iron transport across the outer membrane [7], loss of this function due to the observed mutation may have caused reduced fitness of the A. xylosoxidans mutant Strain 4. However, from a clinical point-of-view, one cannot exclude a spontaneous improvement, independently of phage therapy and despite months of fruitless antibiotic treatment, highlighting the dire need of randomized clinical trials in order to more convincingly assess the clinical efficacy of bacteriophages. In conclusion, we describe the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. Despite initial persisting airway colonization, the final clinical and microbiological outcome was favorable. Whole genome sequencing of eight A. xylosoxidans isolates and strain-specific qPCR experiments performed on BALs sampled before and after phage therapy allowed to more precisely study the dynamics of lung colonization by different isolates, susceptible or resistant to the applied phage cocktails. Acknowledgments The authors would like to thank L. Lécuyer and V. Boussaud (Unité de Transplantation, HEGP), B. Sabatier, V. Savoldelli, T. Caruba and P. Prognon (Pharmacie HEGP), P. Odou, C. Nassar, C. Berneron and M. Roche (Pharmacie Lille), and E. Deberranger (Hématologie Pédiatrique Lille), for their help in the management of the patient. Microbiological analysis was performed with a funding by the Association Robert Debré pour la Recherche Médicale. The authors would also like to thank Caroline Semaille and Nathalie Morgensztejn from the Agence Nationale de Sécurité du Médicament (ANSM) for their regulatory support. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Materials The following are available online at https://www.mdpi.com/1999-4915/13/1/60/s1, Figure S1: Antibiotic susceptibility testing (AST) of a pandrug-resistant Achromobacter xylosoxidans isolated during a bronchoalveolar lavage of a 12-year-old boy after double lung transplantation, Table S1: Variant calling for the eight Achromobacter xylosoxidans whole genome sequences, see excel file attached, named: “Supplementary Table S1. Variant calling.xls”. Click here for additional data file. Author Contributions D.L. (David Lebeaux) and M.V. conceptualized the study, validated the data, carried out formal analysis, prepared the initial draft, took care of project administration and supervised the study. Data were visualized by H.D. and Y.G. Software analysis was carried out by H.D., S.V., Y.G. D.L. (David Lebeaux), M.M., R.G., I.P., F.C., N.K., J.W., C.R., E.C., D.L. (Damien Lanoy), L.V.S., H.D., J.-P.P., N.D., S.V., Y.G., F.V.N., and M.V. contributed to basic methodology. Data curation was done by D.L. (David Lebeaux), M.M., C.T., S.V., Y.G., and M.V. Funding was acquired by D.L. (David Lebeaux), J.-L.M., F.V.N., and M.V. All authors took part in the investigation and in final reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement The study did not require ethical approval. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Phylogenetic tree built with Whole genome sequences of eight Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. The distance between samples reflects the number of different Single Nucleotide Polymorphisms (SNPs). Each A. xylosoxidans is named as follows: isolate number (is1 to is8) S (or R to depict susceptibility or resistance to phage cocktail)-Strain number/Number of BAL where it has been identified. viruses-13-00060-t001_Table 1Table 1 Results of bronchoalveolar lavages (BALs) and sputum analysis from May 2017, to April 2020, in 12-year-old lung transplanted patient who received two rounds of phage therapy for pandrug-resistant Achromobacter xylosoxidans colonization and infection. Date 19 May 2017 23 May 2017 6 June 2017 12 June 2017 25 July 2017 5 September 2017 8 September 2017 15 September 2017 12 October 2017 27 October 2017 23 January 2018 8 February 2018 5 March 2018 18 May 2018 19 February 2019 19 June 2019 27 August 2019 30 April 2020 Phage therapy ROUND 1 ROUND 2 Tigecycline from 29 June to 31 July Imipenem From 31 July 2017, to 16 February 2018 Number of days, relative to the first round of phage therapy −112 −108 −94 −88 −45 −3 +7 +37 +49 +137 +153 +178 +252 +529 +649 +718 +963 BAL BAL Number * #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 Leukocytes (/mm3) >1000 800 >1000 500 >1000 60 >1000 810 >1000 NA NA NA PMN cells (%) 90 88 90 91 43 30 80 69 82 NA NA NA Quantification of A. x culture (CFU/mL) 104 103 104 103 104 103 104 106 105 104 0 0 Other bacteria (CFU/mL) None None None None None None None None None None 104 S. a 106 S. a Sputum Leucocytes (/field) >25 >25 >25 >25 NA NA NA Quantification of A. x culture (CFU/mL) 105 105 105 106 103 103 103 Other bacteria (CFU/mL) None None None None 108 M. c None 106 S. a A. x: Achromobacter xylosoxidans; M. c: Moraxella catarrhalis; NA: non available; PMN: Polymorphonuclear; RBC: red blood cells; S. a: Staphylococcus aureus. : BAL number in bold indicates BALs for which qPCR had been carried out. viruses-13-00060-t002_Table 2Table 2 Eight Achromobacter xylosoxidans isolates for which whole genome sequencing was carried out. These were isolated from bronchoalveolar lavages in a 12-year-old boy after double lung transplantation. The patient received a first round of phage administration on 8 September 2017. A second round of phage treatment was performed on 23 January 2018. Complete Name Isolate Strain Source of Sampling Date of Sampling Whole Genome Sequence is1S-Str1/BAL#3 is1S Str1 BAL#3 25 July 2017 ERS5044236-UGAX1 is2S-Str2/BAL#5 is2S Str2 BAL#5 15 September 2017 ERS5044237-UGAX2 is3R-Str1/BAL#5 is3R Str1 BAL#5 15 September 2017 ERS5044238-UGAX3 is5S-Str3/BAL#6 is5S Str3 BAL#6 12 October 2017 ERS5044239-UGAX5 is6S-Str3/BAL#6 is6S Str3 BAL#6 12 October 2017 ERS5044240-UGAX6 is7R-Str4/BAL#9 is7R Str4 BAL#9 8 February 2018 ERS5044241-UGAX7 is8R-Str4/BAL#9 is8R Str4 BAL#9 8 February 2018 ERS5044242-UGAX8 is9S-Str3/BAL#9 is9S Str3 BAL#9 8 February 2018 ERS5044243-UGAX9 * Submitted at ENA, 1 September 2020. Study ID: PRJEB39103. BAL: Bronchoalveolar lavage, S: Phage susceptible, R: phage resistant. viruses-13-00060-t003_Table 3Table 3 Results of bacterial culture (grey-shading) and qPCR of three broncho-alveolar lavages for four different Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. Bronchoalveolar Lavage BAL#5 BAL#6 BAL#9 15 September 2017 12 October 2017 8 February 2018 qPCR for Strain Isolates Str1 is1S, is3R N NS P (34.7) Str2 is2S P (36.6) N N Str3 is5S, is6S, is9S P (32.7) P (28.9) P (27.7) Str4 is7R, is8R NS NS P (35.2) Grey-shaded box = isolate cultured from this BAL; N: negative (Cq-value > 40); NS: Nonspecific amplification (melting peak different from that of specific amplification); P: positive qPCR (Cq-value).	Recovered	ReactionOutcome	CC BY	33466377	19,957,163	2021-01-05
What was the outcome of reaction 'Pathogen resistance'?	A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient. Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains. 1. Case Presentation We report the case of a 12-year-old boy (35.5 kg), who received double lung transplantation in 25 March 2017, (at the age of 12) because of cystic fibrosis. Before transplantation, he was colonized with Aspergillus fumigatus, Achromobacter xylosoxidans (first colonization identified in 2013), and Pseudomonas aeruginosa. After transplantation, he experienced acute kidney injury requiring temporary haemodialysis, pulmonary embolism, and persisting airway colonization with A. fumigatus. He was discharged on 29 April 2017, with satisfactory respiratory status; he initially required no home oxygen therapy. His immunosuppressive regimen included prednisolone (25 mg/day), tacrolimus (1.5 mg bis in die (b.i.d.) with a target trough concentration of 6–8 ng/mL), mycophenolate mofetil (1 g b.i.d.) and his antimicrobial prophylaxis was cotrimoxazole (400/80 mg, three times a week), posaconazole (150 mg, oral suspension, ter in die (t.i.d.)), valacyclovir (500 mg b.i.d.), and intravenous immunoglobulins (15 g each three weeks). Between May and June 2017, he experienced progressive shortness of breath, cough and increased sputum production; he subsequently required the addition of 1–1.5 L/min oxygen therapy. Because of a stenosis of the surgical anastomosis of the main left bronchus, a dilatation during rigid bronchoscopy was performed on 19 May and a Montgomery stent was inserted on 2 June. Later on, a stenosis of the surgical anastomosis on the right bronchus intermedius required a dilatation during rigid bronchoscopy followed by the insertion of an Oki stent on 29 June. Several bronchoalveolar lavages (BAL), performed between May and June 2017, revealed inflammation (between 800 and >1000 leukocytes/mm3 with 88–90% of polymorphonuclear cells) and repeatedly grew pandrug-resistant A. xylosoxidans (Table 1 and Supplementary Figure S1) [1]. Lung biopsy performed on 19 June was consistent with lung infection: diffuse acute bronchiolitis with extension to peri-bronchiolar alveoli. Microbiological samples obtained from lung biopsies were negative (bacterial, mycological, and mycobacteriological cultures). The first antibiotic treatment consisted of intravenous tigecycline 50 mg b.i.d. from 29 June to 31 July. As BAL remained positive for A. xylosoxidans and the patient still required oxygen therapy; antibiotic treatment was subsequently switched to imipenem (1200 mg, t.i.d., ~100 mg/Kg/d) on 31 July 2017. Subsequent respiratory samples still grew A. xylosoxidans and the respiratory status did not improve (requiring oxygen at home, 1 L/min). Furthermore, despite antibiotic treatment and correct drainage of the right superior lobe, lung consolidation and micronodules remained. Considering the failure of antibiotic treatment, phage therapy was proposed to the patient and his family, after multidisciplinary discussion. A first cocktail (APC 1.1) containing three lytic phages (JWDelta, JWT and 2-1) active against A. xylosoxidans isolate is1S (designated such as later), selected from the DSMZ collection (Braunschweig, Germany) [2,3], was prepared at the Laboratory for Bacteriology Research (Ghent University, Belgium) and the Laboratory for Molecular and Cellular Technology (Queen Astrid Military Hospital, Brussels, Belgium) using the above-mentioned bacterial isolate, as previously described [4]. The production was completed on 23 August with a final phage titer of 4 × 1010 plaque forming units (pfu)/mL defined by double-agar overlay method [4], an endotoxin level of 4000 EU/mL (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, GenScript, Piscataway Township, NJ, USA) and a pH of 7.3 (in sterile PBS). Regulatory permission for phage importation was obtained on 1 September 2017, from the Agence Nationale de Sécurité du Médicament (ANSM). A first round of phage administration was performed, consisting of 3 nebulizations/day of 5 mL of the filter-sterilized (Sartorius 0.2 µm) phage solution, tenfold diluted in sterile saline on 8 and 9 September, using a vibrating mesh nebulizer (eFLOW rapid, PARI, PARI GmbH, Germany) with a mouth piece. Immediate tolerance was perfect, but no clinical improvement was noted and culture of subsequent respiratory samples remained positive for A. xylosoxidans (104 CFU/mL in the BAL of 15 September). A second cocktail (APC 2.1) was produced, in which phage JWalpha was added to the three lytic phages of cocktail APC 1.1, to improve the therapeutic efficacy. The production was completed on 22 December with a final phage titer of 5 × 109 pfu/mL, an endotoxin level of 1760 EU/mL and a pH of 6.95 (in sterile PBS). Regulatory permission was obtained for phage importation on 16 January 2018 (ANSM). On 23 January, during therapeutic bronchoscopy under general anesthesia, 30 mL of APC2.1, tenfold diluted (in sterile saline) and filter-sterilized, was instilled in each pulmonary lobe through the fibroscope. Immediate tolerance was perfect and the patient was discharged on 24 January with continued phage nebulization at home: three times a day 5 mL of preparation A using a vibrating mesh nebulizer (eFLOW rapid, PARI) with a mouth piece for 14 days until 6 February. Clinical tolerance was perfect again, but the initial clinical status remained unchanged. BAL performed on 8 February 2018, two days after stopping the phage treatment, still grew 105 cfu/mL of A. xylosoxidans. Subsequently, the patient’s respiratory condition slowly improved and oxygen therapy was stopped on 15 February 2018. No clinical worsening was observed after imipenem interruption on 16 February 2018. Sputum culture remained positive for A. xylosoxidans but with a low bacterial density (103 CFU/mL in March and May 2018, and June 2019) and no A. xylosoxidans could be isolated from BALs#10 and #11, sampled on August 2019, and April 2020, respectively (Table 1). The last pulmonary function tests, performed on 17 October 2019, showed best results since lung transplantation, with an FEV1 of 79% and a forced vital capacity of 85%. 2. Microbiological Analysis of Eight A. xylosoxidans Isolates As we observed discrepant outcome in our patient (clinical improvement on the one hand but persisting low-grade airway colonization on the other hand), we decided to study the eight available A. xylosoxidans isolates (is1 to is9) from the patient’s colonization obtained by random picking from the culture plates from 4 distinct BALs (Table 2). These eight isolates, taken before, during and after the phage therapy period (Table 2), could not be differentiated from each other by means of MALDI-TOF [5] or McRAPD typing [6] and all belonged to the epidemic clone, already described previously among Belgian CF patients [5]. Whole genome sequencing (WGS) and de novo genome assembly for these eight isolates was carried out (Supplementary Methods and Table S1). The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Total genome length for all eight isolates was 6.44–6.50 Mbp, with a GC content of 67.5%. All eight isolates showed most similarity with reference genome GCF_008432465 (AX1), which corresponds to the type strain of A. xylosoxidans subsp. xylosoxidans (ATCC 27061T, CCUG 12689T, DSMZ 10346T, LMG 1863T, and NCTC 10807T), i.e., between 88.38 and 89.60% on the basis of mapped sequence reads, and between 98.71% and 98.76% on the basis of average nucleotide identity (ANI). Indeed, the overall similarity between the genome sequences of the eight isolates was high, i.e., 99.60%, as assessed by digital DNA hybridization, and more than 99.97% according to ANI-analysis. This was confirmed by variant calling where we observed that the isolates differed from each other only for a total of 367 SNPs, whereas a total of 59,452 SNPs were observed between the isolates and the best matching reference genome (AX1) (Figure 1). The susceptibility phenotype of each of the eight isolates was assessed by spot-test against the phage cocktail APC 2.1 and expressed as “S” when susceptible or “R” when resistant (e.g., “is2S” means that isolate number 2 was susceptible to the phage cocktail) (Table 2 and Supplementary Methods). Among these eight isolates, analysis based on variant calling furthermore revealed four closely related but clearly delineated strains, i.e., clones that differ genetically from other clones, with different phage susceptibility phenotype (Table 2 and Figure 1): Strain 1 (Str1), contained isolates 1 and 3 (is1S and is3R), found in BAL#3 (is1S-Str1/BAL#3) and BAL#5 (is3R-Str1/BAL#5), respectively. Strain 2 (Str2) contained isolate 2 (is2S) from BAL#5 (is2S-Str2/BAL#5). Strain 3 (Str3) contained isolates 5, 6, and 9 (is5S, is6S and is9S), which were found in BAL#6 (is5S-Str3/BAL#6, is6S-Str3/BAL#6) and BAL#9 (is9S-Str3/BAL#9). Strain 4 contained isolates 7 and 8 (is7R and is8R), found in BAL#9 (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) (Table 2). Isolate is1S-Str1/BAL#3 was obtained on 25 July 2017, i.e., 45 days before the first phage therapy session (8 September 2017) and was 100% identical to is3R-Str1/BAL#5 isolated seven days (15 September 2017) after the first phage therapy session (Figure 1). Since both isolates is1S-Str1/BAL#3 and is3R-Str1/BAL#5 are 100% identical, this means that the phage resistance mechanism of isolate is3R-Str1/BAL#5 is not genetically supported (see discussion). Isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6, both phage susceptible, were obtained one month later (12 October 2017) and belonged to still another strain (Strain 3). Finally, on 8 February 2018, i.e., 16 days after the second phage therapy session (23 January 2018), an additional three isolates were obtained from BAL#9. Of these, isolate is9S-Str3/BAL#9 belonged to the same Strain 3 as isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6 and was phage susceptible as well, but isolates 7R and 8R belonged to still another strain (Strain 4) and were both resistant to the phage cocktail. Interestingly, the phage resistant Strain 4 isolates (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) contained mutations compared to the other six isolates that could explain the phage resistance observed for these two isolates (Figure 1): when Strain 1 was used as a reference, a C to A transition at position 1803 of the colicin I receptor gene caused a Tyr601->Stop transition. This receptor has been recognized as a phage receptor [7]. We further studied the course of infection by extracting DNA from the three available A. xylosoxidans-positive BAL samples (BAL#5, #6 and #9), and by quantifying DNA of the different strains by means of strain-specific qPCRs (Table 3 and Supplementary Methods) that had been developed on the basis of the obtained WGS data. qPCRs for Strain 2 and Strain 4 were only positive for the BALs from which these isolates had been cultured, respectively, BAL#5 and BAL#9. qPCR for Strain 1 was only positive for BAL#9, but not for BAL#5 from which isolate 3R had been cultured. qPCR for Strain 3 was positive for all three BALs, although no isolate belonging to Strain 3 was cultured from BAL#5. 3. Discussion and Conclusions We report the first case of decolonization of pandrug-resistant Achromobacter xylosoxidans after a long-term follow-up in a lung-transplanted patient receiving phage therapy. The first actual demonstration and usage of bacteriophages came almost simultaneously from two microbiologists in 1915 (Frederick Twort) and 1917 (Félix d’Hérelle) [8]. After extensive clinical use during the first half of the 20th century, their use has been progressively reduced, mainly due to the discovery, rapid spread, and ease of use of antibiotics for the treatment of infectious diseases [9]. However, bacteriophages gained a new surge of attention, due to the emergence and spread of pandrug-resistant bacteria [8]. Despite decades of clinical experience with bacteriophages in Eastern Europe, very few data are available regarding their clinical efficacy; besides, case-series and few randomized controlled trials (RCT) [10,11,12]. For instance, a RCT assessed the clinical outcome of patients receiving local application of a phage cocktail for the treatment of burn wound infection. This RCT is frequently cited to have shown no clinical benefits, as compared to standard of care. However, the phage titer that was applied (100 pfu/mL) [10], was so extremely low that it precludes to draw any conclusion from this study regarding the usefulness of phage therapy. The limited number of randomized clinical trials has been recently reviewed [13]. Phage therapy has also been used locally, e.g., for the treatment of bone and joint infections [14,15] or intravenously for the treatment of bloodstream infections [16,17]. Considering the high frequency of colonization and infection caused by antibiotic-resistant bacteria among cystic fibrosis and lung-transplanted patients, such as in our patient, the use of bacteriophages appears to be an appealing option in this population [18,19,20]. In the specific case of A. xylosoxidans, thus far a single case-report described the favorable outcome after the use of bacteriophages in a cystic fibrosis patient [21]. Our case-report highlights the many difficulties that need to be taken into account before considering the use of bacteriophages in a clinical setting. These include amongst others an important delay from clinical decision to regulatory agency authorization. Most problematic is the difficult interpretation of the clinical outcome after bacteriophage therapy, considering the long-term persisting airway colonization and the possible evolution of different A. xylosoxidans strains. The second therapeutic attempt relied on a massive lung instillation (injection of the bacteriophage solution in the patient’s lung during fibroscopy) followed by a two-week course of phage nebulization. We used a vibrating mesh nebulizer for phage delivery as this type of nebulizer has been shown to result in a lower phage titer reduction, as compared to jet nebulizers [22]. BAL#9, performed two weeks after the second round of phage therapy (8 February 2018) still grew 105 CFU/mL of A. xylosoxidans, represented by Strains 3 and 4 (according to culture) and by at least Strains 1, 3 and 4 (according to qPCR) (Table 1, Table 2 and Table 3). Sputum cultures remained positive for 18 months, but with only 103 CFU A. xylosoxidans/mL, suggesting a possible upper airway colonization, especially since no A. xylosoxidans could be isolated from BALs sampled in August 2019, and April 2020. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. To more precisely understand the discrepant outcome of our patient, another option would have been to search for active phages from the BAL samples throughout the study, but this was not performed. Despite the observed differences of a total of 367 SNPs between the four strains of eight A. xylosoxidans isolates from the patient, their overall similarity suggests that they originate from a single strain that colonized the patient (who was colonized with A. xylosoxidans years before the start of the phage therapy). Therefore, it is possible that some variants were present already before lung transplantation and before the start of phage therapy. Although it is not possible to exclude the simultaneous presence of these different strains on the basis of our culture results, because only one or two colonies were picked from each BAL culture, analysis by means of four strain-specific qPCR assays of the three BAL samples that were available for study, indicated that Strains 2 and 4 were probably prevalent at a particular moment of the course of infection, whereas Strains 1 and 3 could be identified during the full course of the infection. Phage resistant Strain 4 may have been selected during phage therapy. Phage training has been proposed to overcome phage resistance and increase infectivity against a given bacterial population [23]. However, training phages may take weeks, and therefore might be restricted to the treatment of chronic infections. Regarding more acute infections, the establishment of phage collections (such as the DSMZ, which provided bacteriophages for our patient) is of critical importance to provide phages active against a broad spectrum of bacterial isolates. An interesting point is the observation of two isolates from the same strain (is1S-Str1/BAL#3 and is3R-Str1/BAL#5) that differ regarding their phage susceptibility phenotype despite the absence of genetic support (identical genome). As observed with bacteria where emerging data point out that a resistance phenotype against a given antimicrobial may not be supported by a genotypic substratum [24,25], phage resistance without any change in genotype has also been documented [26]. These genotype/phenotype discrepancies are hypothesized to be possible through epigenetic regulation mechanisms, of which post-transcriptional regulation, which may influence phage receptor expression on the cell surface or phage receptor masking (through capsule synthesis). Further studies are required to better understand the dynamics of in vivo selection of phage-resistant bacteria. Because the patient’s medical condition improved anyhow, a few weeks after the second round of phage instillation, it is possible that phage therapy selected the phage-resistant A. xylosoxidans Strain 4, but that the mutation that caused phage resistance at the same time decreased the virulence of the strain as already described with other bacteria and phages [27,28,29]. Indeed, we observed several mutations which could explain the phage resistance of Strain 4, of which one was especially interesting, since it caused the gain of a stop codon in the colicin I receptor, which has been recognized as a phage receptor in other species [7]. Because in E. coli, the colicin I receptor plays a crucial role in iron transport across the outer membrane [7], loss of this function due to the observed mutation may have caused reduced fitness of the A. xylosoxidans mutant Strain 4. However, from a clinical point-of-view, one cannot exclude a spontaneous improvement, independently of phage therapy and despite months of fruitless antibiotic treatment, highlighting the dire need of randomized clinical trials in order to more convincingly assess the clinical efficacy of bacteriophages. In conclusion, we describe the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. Despite initial persisting airway colonization, the final clinical and microbiological outcome was favorable. Whole genome sequencing of eight A. xylosoxidans isolates and strain-specific qPCR experiments performed on BALs sampled before and after phage therapy allowed to more precisely study the dynamics of lung colonization by different isolates, susceptible or resistant to the applied phage cocktails. Acknowledgments The authors would like to thank L. Lécuyer and V. Boussaud (Unité de Transplantation, HEGP), B. Sabatier, V. Savoldelli, T. Caruba and P. Prognon (Pharmacie HEGP), P. Odou, C. Nassar, C. Berneron and M. Roche (Pharmacie Lille), and E. Deberranger (Hématologie Pédiatrique Lille), for their help in the management of the patient. Microbiological analysis was performed with a funding by the Association Robert Debré pour la Recherche Médicale. The authors would also like to thank Caroline Semaille and Nathalie Morgensztejn from the Agence Nationale de Sécurité du Médicament (ANSM) for their regulatory support. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Materials The following are available online at https://www.mdpi.com/1999-4915/13/1/60/s1, Figure S1: Antibiotic susceptibility testing (AST) of a pandrug-resistant Achromobacter xylosoxidans isolated during a bronchoalveolar lavage of a 12-year-old boy after double lung transplantation, Table S1: Variant calling for the eight Achromobacter xylosoxidans whole genome sequences, see excel file attached, named: “Supplementary Table S1. Variant calling.xls”. Click here for additional data file. Author Contributions D.L. (David Lebeaux) and M.V. conceptualized the study, validated the data, carried out formal analysis, prepared the initial draft, took care of project administration and supervised the study. Data were visualized by H.D. and Y.G. Software analysis was carried out by H.D., S.V., Y.G. D.L. (David Lebeaux), M.M., R.G., I.P., F.C., N.K., J.W., C.R., E.C., D.L. (Damien Lanoy), L.V.S., H.D., J.-P.P., N.D., S.V., Y.G., F.V.N., and M.V. contributed to basic methodology. Data curation was done by D.L. (David Lebeaux), M.M., C.T., S.V., Y.G., and M.V. Funding was acquired by D.L. (David Lebeaux), J.-L.M., F.V.N., and M.V. All authors took part in the investigation and in final reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement The study did not require ethical approval. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Phylogenetic tree built with Whole genome sequences of eight Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. The distance between samples reflects the number of different Single Nucleotide Polymorphisms (SNPs). Each A. xylosoxidans is named as follows: isolate number (is1 to is8) S (or R to depict susceptibility or resistance to phage cocktail)-Strain number/Number of BAL where it has been identified. viruses-13-00060-t001_Table 1Table 1 Results of bronchoalveolar lavages (BALs) and sputum analysis from May 2017, to April 2020, in 12-year-old lung transplanted patient who received two rounds of phage therapy for pandrug-resistant Achromobacter xylosoxidans colonization and infection. Date 19 May 2017 23 May 2017 6 June 2017 12 June 2017 25 July 2017 5 September 2017 8 September 2017 15 September 2017 12 October 2017 27 October 2017 23 January 2018 8 February 2018 5 March 2018 18 May 2018 19 February 2019 19 June 2019 27 August 2019 30 April 2020 Phage therapy ROUND 1 ROUND 2 Tigecycline from 29 June to 31 July Imipenem From 31 July 2017, to 16 February 2018 Number of days, relative to the first round of phage therapy −112 −108 −94 −88 −45 −3 +7 +37 +49 +137 +153 +178 +252 +529 +649 +718 +963 BAL BAL Number * #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 Leukocytes (/mm3) >1000 800 >1000 500 >1000 60 >1000 810 >1000 NA NA NA PMN cells (%) 90 88 90 91 43 30 80 69 82 NA NA NA Quantification of A. x culture (CFU/mL) 104 103 104 103 104 103 104 106 105 104 0 0 Other bacteria (CFU/mL) None None None None None None None None None None 104 S. a 106 S. a Sputum Leucocytes (/field) >25 >25 >25 >25 NA NA NA Quantification of A. x culture (CFU/mL) 105 105 105 106 103 103 103 Other bacteria (CFU/mL) None None None None 108 M. c None 106 S. a A. x: Achromobacter xylosoxidans; M. c: Moraxella catarrhalis; NA: non available; PMN: Polymorphonuclear; RBC: red blood cells; S. a: Staphylococcus aureus. : BAL number in bold indicates BALs for which qPCR had been carried out. viruses-13-00060-t002_Table 2Table 2 Eight Achromobacter xylosoxidans isolates for which whole genome sequencing was carried out. These were isolated from bronchoalveolar lavages in a 12-year-old boy after double lung transplantation. The patient received a first round of phage administration on 8 September 2017. A second round of phage treatment was performed on 23 January 2018. Complete Name Isolate Strain Source of Sampling Date of Sampling Whole Genome Sequence is1S-Str1/BAL#3 is1S Str1 BAL#3 25 July 2017 ERS5044236-UGAX1 is2S-Str2/BAL#5 is2S Str2 BAL#5 15 September 2017 ERS5044237-UGAX2 is3R-Str1/BAL#5 is3R Str1 BAL#5 15 September 2017 ERS5044238-UGAX3 is5S-Str3/BAL#6 is5S Str3 BAL#6 12 October 2017 ERS5044239-UGAX5 is6S-Str3/BAL#6 is6S Str3 BAL#6 12 October 2017 ERS5044240-UGAX6 is7R-Str4/BAL#9 is7R Str4 BAL#9 8 February 2018 ERS5044241-UGAX7 is8R-Str4/BAL#9 is8R Str4 BAL#9 8 February 2018 ERS5044242-UGAX8 is9S-Str3/BAL#9 is9S Str3 BAL#9 8 February 2018 ERS5044243-UGAX9 * Submitted at ENA, 1 September 2020. Study ID: PRJEB39103. BAL: Bronchoalveolar lavage, S: Phage susceptible, R: phage resistant. viruses-13-00060-t003_Table 3Table 3 Results of bacterial culture (grey-shading) and qPCR of three broncho-alveolar lavages for four different Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. Bronchoalveolar Lavage BAL#5 BAL#6 BAL#9 15 September 2017 12 October 2017 8 February 2018 qPCR for Strain Isolates Str1 is1S, is3R N NS P (34.7) Str2 is2S P (36.6) N N Str3 is5S, is6S, is9S P (32.7) P (28.9) P (27.7) Str4 is7R, is8R NS NS P (35.2) Grey-shaded box = isolate cultured from this BAL; N: negative (Cq-value > 40); NS: Nonspecific amplification (melting peak different from that of specific amplification); P: positive qPCR (Cq-value).	Recovered	ReactionOutcome	CC BY	33466377	19,957,163	2021-01-05
What was the outcome of reaction 'Pneumonia bacterial'?	A Case of Phage Therapy against Pandrug-Resistant Achromobacter xylosoxidans in a 12-Year-Old Lung-Transplanted Cystic Fibrosis Patient. Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains. 1. Case Presentation We report the case of a 12-year-old boy (35.5 kg), who received double lung transplantation in 25 March 2017, (at the age of 12) because of cystic fibrosis. Before transplantation, he was colonized with Aspergillus fumigatus, Achromobacter xylosoxidans (first colonization identified in 2013), and Pseudomonas aeruginosa. After transplantation, he experienced acute kidney injury requiring temporary haemodialysis, pulmonary embolism, and persisting airway colonization with A. fumigatus. He was discharged on 29 April 2017, with satisfactory respiratory status; he initially required no home oxygen therapy. His immunosuppressive regimen included prednisolone (25 mg/day), tacrolimus (1.5 mg bis in die (b.i.d.) with a target trough concentration of 6–8 ng/mL), mycophenolate mofetil (1 g b.i.d.) and his antimicrobial prophylaxis was cotrimoxazole (400/80 mg, three times a week), posaconazole (150 mg, oral suspension, ter in die (t.i.d.)), valacyclovir (500 mg b.i.d.), and intravenous immunoglobulins (15 g each three weeks). Between May and June 2017, he experienced progressive shortness of breath, cough and increased sputum production; he subsequently required the addition of 1–1.5 L/min oxygen therapy. Because of a stenosis of the surgical anastomosis of the main left bronchus, a dilatation during rigid bronchoscopy was performed on 19 May and a Montgomery stent was inserted on 2 June. Later on, a stenosis of the surgical anastomosis on the right bronchus intermedius required a dilatation during rigid bronchoscopy followed by the insertion of an Oki stent on 29 June. Several bronchoalveolar lavages (BAL), performed between May and June 2017, revealed inflammation (between 800 and >1000 leukocytes/mm3 with 88–90% of polymorphonuclear cells) and repeatedly grew pandrug-resistant A. xylosoxidans (Table 1 and Supplementary Figure S1) [1]. Lung biopsy performed on 19 June was consistent with lung infection: diffuse acute bronchiolitis with extension to peri-bronchiolar alveoli. Microbiological samples obtained from lung biopsies were negative (bacterial, mycological, and mycobacteriological cultures). The first antibiotic treatment consisted of intravenous tigecycline 50 mg b.i.d. from 29 June to 31 July. As BAL remained positive for A. xylosoxidans and the patient still required oxygen therapy; antibiotic treatment was subsequently switched to imipenem (1200 mg, t.i.d., ~100 mg/Kg/d) on 31 July 2017. Subsequent respiratory samples still grew A. xylosoxidans and the respiratory status did not improve (requiring oxygen at home, 1 L/min). Furthermore, despite antibiotic treatment and correct drainage of the right superior lobe, lung consolidation and micronodules remained. Considering the failure of antibiotic treatment, phage therapy was proposed to the patient and his family, after multidisciplinary discussion. A first cocktail (APC 1.1) containing three lytic phages (JWDelta, JWT and 2-1) active against A. xylosoxidans isolate is1S (designated such as later), selected from the DSMZ collection (Braunschweig, Germany) [2,3], was prepared at the Laboratory for Bacteriology Research (Ghent University, Belgium) and the Laboratory for Molecular and Cellular Technology (Queen Astrid Military Hospital, Brussels, Belgium) using the above-mentioned bacterial isolate, as previously described [4]. The production was completed on 23 August with a final phage titer of 4 × 1010 plaque forming units (pfu)/mL defined by double-agar overlay method [4], an endotoxin level of 4000 EU/mL (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, GenScript, Piscataway Township, NJ, USA) and a pH of 7.3 (in sterile PBS). Regulatory permission for phage importation was obtained on 1 September 2017, from the Agence Nationale de Sécurité du Médicament (ANSM). A first round of phage administration was performed, consisting of 3 nebulizations/day of 5 mL of the filter-sterilized (Sartorius 0.2 µm) phage solution, tenfold diluted in sterile saline on 8 and 9 September, using a vibrating mesh nebulizer (eFLOW rapid, PARI, PARI GmbH, Germany) with a mouth piece. Immediate tolerance was perfect, but no clinical improvement was noted and culture of subsequent respiratory samples remained positive for A. xylosoxidans (104 CFU/mL in the BAL of 15 September). A second cocktail (APC 2.1) was produced, in which phage JWalpha was added to the three lytic phages of cocktail APC 1.1, to improve the therapeutic efficacy. The production was completed on 22 December with a final phage titer of 5 × 109 pfu/mL, an endotoxin level of 1760 EU/mL and a pH of 6.95 (in sterile PBS). Regulatory permission was obtained for phage importation on 16 January 2018 (ANSM). On 23 January, during therapeutic bronchoscopy under general anesthesia, 30 mL of APC2.1, tenfold diluted (in sterile saline) and filter-sterilized, was instilled in each pulmonary lobe through the fibroscope. Immediate tolerance was perfect and the patient was discharged on 24 January with continued phage nebulization at home: three times a day 5 mL of preparation A using a vibrating mesh nebulizer (eFLOW rapid, PARI) with a mouth piece for 14 days until 6 February. Clinical tolerance was perfect again, but the initial clinical status remained unchanged. BAL performed on 8 February 2018, two days after stopping the phage treatment, still grew 105 cfu/mL of A. xylosoxidans. Subsequently, the patient’s respiratory condition slowly improved and oxygen therapy was stopped on 15 February 2018. No clinical worsening was observed after imipenem interruption on 16 February 2018. Sputum culture remained positive for A. xylosoxidans but with a low bacterial density (103 CFU/mL in March and May 2018, and June 2019) and no A. xylosoxidans could be isolated from BALs#10 and #11, sampled on August 2019, and April 2020, respectively (Table 1). The last pulmonary function tests, performed on 17 October 2019, showed best results since lung transplantation, with an FEV1 of 79% and a forced vital capacity of 85%. 2. Microbiological Analysis of Eight A. xylosoxidans Isolates As we observed discrepant outcome in our patient (clinical improvement on the one hand but persisting low-grade airway colonization on the other hand), we decided to study the eight available A. xylosoxidans isolates (is1 to is9) from the patient’s colonization obtained by random picking from the culture plates from 4 distinct BALs (Table 2). These eight isolates, taken before, during and after the phage therapy period (Table 2), could not be differentiated from each other by means of MALDI-TOF [5] or McRAPD typing [6] and all belonged to the epidemic clone, already described previously among Belgian CF patients [5]. Whole genome sequencing (WGS) and de novo genome assembly for these eight isolates was carried out (Supplementary Methods and Table S1). The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Total genome length for all eight isolates was 6.44–6.50 Mbp, with a GC content of 67.5%. All eight isolates showed most similarity with reference genome GCF_008432465 (AX1), which corresponds to the type strain of A. xylosoxidans subsp. xylosoxidans (ATCC 27061T, CCUG 12689T, DSMZ 10346T, LMG 1863T, and NCTC 10807T), i.e., between 88.38 and 89.60% on the basis of mapped sequence reads, and between 98.71% and 98.76% on the basis of average nucleotide identity (ANI). Indeed, the overall similarity between the genome sequences of the eight isolates was high, i.e., 99.60%, as assessed by digital DNA hybridization, and more than 99.97% according to ANI-analysis. This was confirmed by variant calling where we observed that the isolates differed from each other only for a total of 367 SNPs, whereas a total of 59,452 SNPs were observed between the isolates and the best matching reference genome (AX1) (Figure 1). The susceptibility phenotype of each of the eight isolates was assessed by spot-test against the phage cocktail APC 2.1 and expressed as “S” when susceptible or “R” when resistant (e.g., “is2S” means that isolate number 2 was susceptible to the phage cocktail) (Table 2 and Supplementary Methods). Among these eight isolates, analysis based on variant calling furthermore revealed four closely related but clearly delineated strains, i.e., clones that differ genetically from other clones, with different phage susceptibility phenotype (Table 2 and Figure 1): Strain 1 (Str1), contained isolates 1 and 3 (is1S and is3R), found in BAL#3 (is1S-Str1/BAL#3) and BAL#5 (is3R-Str1/BAL#5), respectively. Strain 2 (Str2) contained isolate 2 (is2S) from BAL#5 (is2S-Str2/BAL#5). Strain 3 (Str3) contained isolates 5, 6, and 9 (is5S, is6S and is9S), which were found in BAL#6 (is5S-Str3/BAL#6, is6S-Str3/BAL#6) and BAL#9 (is9S-Str3/BAL#9). Strain 4 contained isolates 7 and 8 (is7R and is8R), found in BAL#9 (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) (Table 2). Isolate is1S-Str1/BAL#3 was obtained on 25 July 2017, i.e., 45 days before the first phage therapy session (8 September 2017) and was 100% identical to is3R-Str1/BAL#5 isolated seven days (15 September 2017) after the first phage therapy session (Figure 1). Since both isolates is1S-Str1/BAL#3 and is3R-Str1/BAL#5 are 100% identical, this means that the phage resistance mechanism of isolate is3R-Str1/BAL#5 is not genetically supported (see discussion). Isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6, both phage susceptible, were obtained one month later (12 October 2017) and belonged to still another strain (Strain 3). Finally, on 8 February 2018, i.e., 16 days after the second phage therapy session (23 January 2018), an additional three isolates were obtained from BAL#9. Of these, isolate is9S-Str3/BAL#9 belonged to the same Strain 3 as isolates is5S-Str3/BAL#6 and is6S-Str3/BAL#6 and was phage susceptible as well, but isolates 7R and 8R belonged to still another strain (Strain 4) and were both resistant to the phage cocktail. Interestingly, the phage resistant Strain 4 isolates (is7R-Str4/BAL#9 and is8R-Str4/BAL#9) contained mutations compared to the other six isolates that could explain the phage resistance observed for these two isolates (Figure 1): when Strain 1 was used as a reference, a C to A transition at position 1803 of the colicin I receptor gene caused a Tyr601->Stop transition. This receptor has been recognized as a phage receptor [7]. We further studied the course of infection by extracting DNA from the three available A. xylosoxidans-positive BAL samples (BAL#5, #6 and #9), and by quantifying DNA of the different strains by means of strain-specific qPCRs (Table 3 and Supplementary Methods) that had been developed on the basis of the obtained WGS data. qPCRs for Strain 2 and Strain 4 were only positive for the BALs from which these isolates had been cultured, respectively, BAL#5 and BAL#9. qPCR for Strain 1 was only positive for BAL#9, but not for BAL#5 from which isolate 3R had been cultured. qPCR for Strain 3 was positive for all three BALs, although no isolate belonging to Strain 3 was cultured from BAL#5. 3. Discussion and Conclusions We report the first case of decolonization of pandrug-resistant Achromobacter xylosoxidans after a long-term follow-up in a lung-transplanted patient receiving phage therapy. The first actual demonstration and usage of bacteriophages came almost simultaneously from two microbiologists in 1915 (Frederick Twort) and 1917 (Félix d’Hérelle) [8]. After extensive clinical use during the first half of the 20th century, their use has been progressively reduced, mainly due to the discovery, rapid spread, and ease of use of antibiotics for the treatment of infectious diseases [9]. However, bacteriophages gained a new surge of attention, due to the emergence and spread of pandrug-resistant bacteria [8]. Despite decades of clinical experience with bacteriophages in Eastern Europe, very few data are available regarding their clinical efficacy; besides, case-series and few randomized controlled trials (RCT) [10,11,12]. For instance, a RCT assessed the clinical outcome of patients receiving local application of a phage cocktail for the treatment of burn wound infection. This RCT is frequently cited to have shown no clinical benefits, as compared to standard of care. However, the phage titer that was applied (100 pfu/mL) [10], was so extremely low that it precludes to draw any conclusion from this study regarding the usefulness of phage therapy. The limited number of randomized clinical trials has been recently reviewed [13]. Phage therapy has also been used locally, e.g., for the treatment of bone and joint infections [14,15] or intravenously for the treatment of bloodstream infections [16,17]. Considering the high frequency of colonization and infection caused by antibiotic-resistant bacteria among cystic fibrosis and lung-transplanted patients, such as in our patient, the use of bacteriophages appears to be an appealing option in this population [18,19,20]. In the specific case of A. xylosoxidans, thus far a single case-report described the favorable outcome after the use of bacteriophages in a cystic fibrosis patient [21]. Our case-report highlights the many difficulties that need to be taken into account before considering the use of bacteriophages in a clinical setting. These include amongst others an important delay from clinical decision to regulatory agency authorization. Most problematic is the difficult interpretation of the clinical outcome after bacteriophage therapy, considering the long-term persisting airway colonization and the possible evolution of different A. xylosoxidans strains. The second therapeutic attempt relied on a massive lung instillation (injection of the bacteriophage solution in the patient’s lung during fibroscopy) followed by a two-week course of phage nebulization. We used a vibrating mesh nebulizer for phage delivery as this type of nebulizer has been shown to result in a lower phage titer reduction, as compared to jet nebulizers [22]. BAL#9, performed two weeks after the second round of phage therapy (8 February 2018) still grew 105 CFU/mL of A. xylosoxidans, represented by Strains 3 and 4 (according to culture) and by at least Strains 1, 3 and 4 (according to qPCR) (Table 1, Table 2 and Table 3). Sputum cultures remained positive for 18 months, but with only 103 CFU A. xylosoxidans/mL, suggesting a possible upper airway colonization, especially since no A. xylosoxidans could be isolated from BALs sampled in August 2019, and April 2020. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. To more precisely understand the discrepant outcome of our patient, another option would have been to search for active phages from the BAL samples throughout the study, but this was not performed. Despite the observed differences of a total of 367 SNPs between the four strains of eight A. xylosoxidans isolates from the patient, their overall similarity suggests that they originate from a single strain that colonized the patient (who was colonized with A. xylosoxidans years before the start of the phage therapy). Therefore, it is possible that some variants were present already before lung transplantation and before the start of phage therapy. Although it is not possible to exclude the simultaneous presence of these different strains on the basis of our culture results, because only one or two colonies were picked from each BAL culture, analysis by means of four strain-specific qPCR assays of the three BAL samples that were available for study, indicated that Strains 2 and 4 were probably prevalent at a particular moment of the course of infection, whereas Strains 1 and 3 could be identified during the full course of the infection. Phage resistant Strain 4 may have been selected during phage therapy. Phage training has been proposed to overcome phage resistance and increase infectivity against a given bacterial population [23]. However, training phages may take weeks, and therefore might be restricted to the treatment of chronic infections. Regarding more acute infections, the establishment of phage collections (such as the DSMZ, which provided bacteriophages for our patient) is of critical importance to provide phages active against a broad spectrum of bacterial isolates. An interesting point is the observation of two isolates from the same strain (is1S-Str1/BAL#3 and is3R-Str1/BAL#5) that differ regarding their phage susceptibility phenotype despite the absence of genetic support (identical genome). As observed with bacteria where emerging data point out that a resistance phenotype against a given antimicrobial may not be supported by a genotypic substratum [24,25], phage resistance without any change in genotype has also been documented [26]. These genotype/phenotype discrepancies are hypothesized to be possible through epigenetic regulation mechanisms, of which post-transcriptional regulation, which may influence phage receptor expression on the cell surface or phage receptor masking (through capsule synthesis). Further studies are required to better understand the dynamics of in vivo selection of phage-resistant bacteria. Because the patient’s medical condition improved anyhow, a few weeks after the second round of phage instillation, it is possible that phage therapy selected the phage-resistant A. xylosoxidans Strain 4, but that the mutation that caused phage resistance at the same time decreased the virulence of the strain as already described with other bacteria and phages [27,28,29]. Indeed, we observed several mutations which could explain the phage resistance of Strain 4, of which one was especially interesting, since it caused the gain of a stop codon in the colicin I receptor, which has been recognized as a phage receptor in other species [7]. Because in E. coli, the colicin I receptor plays a crucial role in iron transport across the outer membrane [7], loss of this function due to the observed mutation may have caused reduced fitness of the A. xylosoxidans mutant Strain 4. However, from a clinical point-of-view, one cannot exclude a spontaneous improvement, independently of phage therapy and despite months of fruitless antibiotic treatment, highlighting the dire need of randomized clinical trials in order to more convincingly assess the clinical efficacy of bacteriophages. In conclusion, we describe the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. Despite initial persisting airway colonization, the final clinical and microbiological outcome was favorable. Whole genome sequencing of eight A. xylosoxidans isolates and strain-specific qPCR experiments performed on BALs sampled before and after phage therapy allowed to more precisely study the dynamics of lung colonization by different isolates, susceptible or resistant to the applied phage cocktails. Acknowledgments The authors would like to thank L. Lécuyer and V. Boussaud (Unité de Transplantation, HEGP), B. Sabatier, V. Savoldelli, T. Caruba and P. Prognon (Pharmacie HEGP), P. Odou, C. Nassar, C. Berneron and M. Roche (Pharmacie Lille), and E. Deberranger (Hématologie Pédiatrique Lille), for their help in the management of the patient. Microbiological analysis was performed with a funding by the Association Robert Debré pour la Recherche Médicale. The authors would also like to thank Caroline Semaille and Nathalie Morgensztejn from the Agence Nationale de Sécurité du Médicament (ANSM) for their regulatory support. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Materials The following are available online at https://www.mdpi.com/1999-4915/13/1/60/s1, Figure S1: Antibiotic susceptibility testing (AST) of a pandrug-resistant Achromobacter xylosoxidans isolated during a bronchoalveolar lavage of a 12-year-old boy after double lung transplantation, Table S1: Variant calling for the eight Achromobacter xylosoxidans whole genome sequences, see excel file attached, named: “Supplementary Table S1. Variant calling.xls”. Click here for additional data file. Author Contributions D.L. (David Lebeaux) and M.V. conceptualized the study, validated the data, carried out formal analysis, prepared the initial draft, took care of project administration and supervised the study. Data were visualized by H.D. and Y.G. Software analysis was carried out by H.D., S.V., Y.G. D.L. (David Lebeaux), M.M., R.G., I.P., F.C., N.K., J.W., C.R., E.C., D.L. (Damien Lanoy), L.V.S., H.D., J.-P.P., N.D., S.V., Y.G., F.V.N., and M.V. contributed to basic methodology. Data curation was done by D.L. (David Lebeaux), M.M., C.T., S.V., Y.G., and M.V. Funding was acquired by D.L. (David Lebeaux), J.-L.M., F.V.N., and M.V. All authors took part in the investigation and in final reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement The study did not require ethical approval. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The sequencing reads and genome sequences of all isolates have been submitted to the European Nucleotide Archive (ENA) and are available under project number PRJEB39103 (Table 2). (https://www.ebi.ac.uk/ena/browser/view/PRJEB39103). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Phylogenetic tree built with Whole genome sequences of eight Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. The distance between samples reflects the number of different Single Nucleotide Polymorphisms (SNPs). Each A. xylosoxidans is named as follows: isolate number (is1 to is8) S (or R to depict susceptibility or resistance to phage cocktail)-Strain number/Number of BAL where it has been identified. viruses-13-00060-t001_Table 1Table 1 Results of bronchoalveolar lavages (BALs) and sputum analysis from May 2017, to April 2020, in 12-year-old lung transplanted patient who received two rounds of phage therapy for pandrug-resistant Achromobacter xylosoxidans colonization and infection. Date 19 May 2017 23 May 2017 6 June 2017 12 June 2017 25 July 2017 5 September 2017 8 September 2017 15 September 2017 12 October 2017 27 October 2017 23 January 2018 8 February 2018 5 March 2018 18 May 2018 19 February 2019 19 June 2019 27 August 2019 30 April 2020 Phage therapy ROUND 1 ROUND 2 Tigecycline from 29 June to 31 July Imipenem From 31 July 2017, to 16 February 2018 Number of days, relative to the first round of phage therapy −112 −108 −94 −88 −45 −3 +7 +37 +49 +137 +153 +178 +252 +529 +649 +718 +963 BAL BAL Number * #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 Leukocytes (/mm3) >1000 800 >1000 500 >1000 60 >1000 810 >1000 NA NA NA PMN cells (%) 90 88 90 91 43 30 80 69 82 NA NA NA Quantification of A. x culture (CFU/mL) 104 103 104 103 104 103 104 106 105 104 0 0 Other bacteria (CFU/mL) None None None None None None None None None None 104 S. a 106 S. a Sputum Leucocytes (/field) >25 >25 >25 >25 NA NA NA Quantification of A. x culture (CFU/mL) 105 105 105 106 103 103 103 Other bacteria (CFU/mL) None None None None 108 M. c None 106 S. a A. x: Achromobacter xylosoxidans; M. c: Moraxella catarrhalis; NA: non available; PMN: Polymorphonuclear; RBC: red blood cells; S. a: Staphylococcus aureus. : BAL number in bold indicates BALs for which qPCR had been carried out. viruses-13-00060-t002_Table 2Table 2 Eight Achromobacter xylosoxidans isolates for which whole genome sequencing was carried out. These were isolated from bronchoalveolar lavages in a 12-year-old boy after double lung transplantation. The patient received a first round of phage administration on 8 September 2017. A second round of phage treatment was performed on 23 January 2018. Complete Name Isolate Strain Source of Sampling Date of Sampling Whole Genome Sequence is1S-Str1/BAL#3 is1S Str1 BAL#3 25 July 2017 ERS5044236-UGAX1 is2S-Str2/BAL#5 is2S Str2 BAL#5 15 September 2017 ERS5044237-UGAX2 is3R-Str1/BAL#5 is3R Str1 BAL#5 15 September 2017 ERS5044238-UGAX3 is5S-Str3/BAL#6 is5S Str3 BAL#6 12 October 2017 ERS5044239-UGAX5 is6S-Str3/BAL#6 is6S Str3 BAL#6 12 October 2017 ERS5044240-UGAX6 is7R-Str4/BAL#9 is7R Str4 BAL#9 8 February 2018 ERS5044241-UGAX7 is8R-Str4/BAL#9 is8R Str4 BAL#9 8 February 2018 ERS5044242-UGAX8 is9S-Str3/BAL#9 is9S Str3 BAL#9 8 February 2018 ERS5044243-UGAX9 * Submitted at ENA, 1 September 2020. Study ID: PRJEB39103. BAL: Bronchoalveolar lavage, S: Phage susceptible, R: phage resistant. viruses-13-00060-t003_Table 3Table 3 Results of bacterial culture (grey-shading) and qPCR of three broncho-alveolar lavages for four different Achromobacter xylosoxidans strains isolated in a 12-year-old boy after double lung transplantation. Bronchoalveolar Lavage BAL#5 BAL#6 BAL#9 15 September 2017 12 October 2017 8 February 2018 qPCR for Strain Isolates Str1 is1S, is3R N NS P (34.7) Str2 is2S P (36.6) N N Str3 is5S, is6S, is9S P (32.7) P (28.9) P (27.7) Str4 is7R, is8R NS NS P (35.2) Grey-shaded box = isolate cultured from this BAL; N: negative (Cq-value > 40); NS: Nonspecific amplification (melting peak different from that of specific amplification); P: positive qPCR (Cq-value).	Recovered	ReactionOutcome	CC BY	33466377	19,957,163	2021-01-05
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Acute kidney injury'.	Prehospital Diagnosis of Shortness of Breath Caused by Profound Metformin-Associated Metabolic Acidosis. Shortness of breath is a common complaint among patients in emergency medicine. While most common causes are usually promptly identified, less frequent aetiologies might be challenging to diagnose, especially in the pre-hospital setting. We report a case of prehospital dyspnoea initially ascribed to pulmonary oedema which turned out to be the result of profound metformin-associated metabolic acidosis. This diagnosis was already made during the prehospital phase by virtue of arterial blood gas measurement. Pre-hospital measurement of arterial blood gases is therefore feasible and can improve diagnostic accuracy in the field, thus avoiding unnecessary delay and potential harm to the patient before initiating the appropriate therapeutic actions. 1. Introduction Shortness of breath (SOB) is one of the most common complaints among patients in prehospital emergency medicine [1]. Most common causes of shortness of breath (pulmonary oedema, pneumonia, chronic obstructive pulmonary disease exacerbation, pulmonary embolism, pneumothorax, etc.) are usually identified rather easily, although less common aetiologies might be overlooked and therefore misdiagnosed if clinicians fail to consider them. Furthermore, even a high clinical suspicion does not equal a definitive diagnosis, which might require para-clinical confirmation through specific investigations such as arterial blood gas (ABG) analysis. We report the case of a patient complaining of SOB that was initially imputed to pulmonary oedema. However, an ABG analysis performed in the field allowed the prehospital emergency physician to identify a profound metformin-associated metabolic acidosis as the actual cause of the patient’s dyspnoea. 2. Case Description The emergency medical call centre (EMCC) dispatched an advanced life support ambulance to take care of a 71-year-old woman who had called complaining about SOB. Upon arrival, prehospital providers noted shallow breathing with a respiratory rate of over 35 breaths per minute. Lung fields were clear to auscultation and the patient showed no sign of cyanosis. There was no haemodynamic compromise. The Glasgow Coma Scale was 15 with no focal deficit and the patient was normothermic. Given the respiratory distress, paramedics initiated oxygen therapy using a high-flow non-rebreather mask. Suspecting acute heart failure (AHF), they asked the EMCC to dispatch a medical mobile unit called ‘Service Mobile d’Urgence et de Réanimation’ (SMUR). These units are staffed by an advanced paramedic and a physician and can be dispatched to assist an ambulance in case of life-threatening emergencies [2]. These SMURs are the only prehospital units equipped with non-invasive ventilation (NIV) devices and portable blood gas analysers (iStat, Abbott Point of Care Inc., Princeton, NJ, USA) and are therefore often called upon to enhance the management of patients in acute respiratory distress or failure. While awaiting the arrival of the SMUR, the paramedics continued their evaluation and discovered that the patient was under metformin for diabetes and diuretics for hypertension, and that she was on day 3 of antibiotic treatment for urinary tract infection. She did not use cigarettes, alcohol, or illicit drugs. The patient was found to be hypoglycaemic at 2.3 mmol/L and received intravenous glucose accordingly. An ABG analysis performed by the SMUR physician shortly after arriving on site showed severe lactic acidosis (FiO2 65%: pH 6.845, PaCO2 1.95 kPa, PaO2 27.7 kPa, HCO3−—2.5 mmol/L, lactate 17.0 mmol/L). While AHF was suspected at first, the profound metabolic acidosis revealed by the ABG analysis was not compatible with the clinical picture. Indeed, the patient was not in cardiogenic shock, and a diagnosis of lactic acidosis secondary to metformin intoxication was thereafter suspected. Intravenous hydration was started and bicarbonates were administered. Since the patient showed signs of respiratory distress and exhaustion, NIV was initiated. In-hospital investigations confirmed severe lactic acidosis and acute kidney injury KDIGO stage 3 [3], with a creatinine of 788 µmol/L which yielded an estimated glomerular filtration rate of 5 mL/min/m2 according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. This kidney injury was secondary to the pre-renal disease on severe dehydration (diuretics, decreased drinking inputs, etc.) and sepsis, with consequent acute tubular necrosis. The patient showed no sign of hepatic impairment nor hypoxaemia. There was no hint that the patient’s illness could have been secondary to suicidal or accidental biguanide ingestion. The diagnosis of profound metformin-associated metabolic acidosis secondary to renal failure was definitively established at this stage. Despite aggressive hydration and bicarbonates infusion, the patient remained oligo-anuric with severe metabolic acidosis and showed signs of respiratory exhaustion finally requiring endotracheal intubation. She was admitted to the intensive care unit (ICU) for haemodynamic and respiratory management. The decision to start haemodialysis was finally made basing on the severity of acidosis and lactate level, coexisting renal failure and failure to improve with bicarbonate therapy [4]. The patient improved rapidly under this treatment and was extubated on the next day after pH normalisation and lactate clearance. She was transferred to the internal medicine ward after 48 h and finally discharged from the hospital 10 days later, after full renal recovery. 3. Discussion SOB is a major complaint among pre-hospital and ED patients, accounting for over 3.9 million visits in 2017 in the United States [5]. Most of these visits involve patients over 65 years old. Generally, cases of SOB are readily identified and can be ascribed to frequent aetiologies, i.e., heart failure (43%), community-acquired pneumonia (35%), acute exacerbation of chronic respiratory disease (32%), pulmonary embolism (18%), and acute asthma (3%). Two coexisting causes of SOB are found in as many as 47% of patients [6]. Nevertheless, clinicians must be aware that other less frequent aetiologies of SOB should be considered (salicylate intoxication, carbon monoxide poisoning, toxin-related metabolic acidosis, diabetic ketoacidosis, sepsis, anaemia, etc.). They should also acknowledge that diagnosing infrequent causes of SOB might be challenging, especially in the pre-hospital setting, where resources are limited and most investigation tools unavailable. Inappropriate dismissal of infrequent causes of SOB might lead to delayed diagnosis and treatment. Such delays might prove harmful, most particularly in old and frail patients. Our case exemplifies how pre-hospital ABG measurement can easily improve diagnostic accuracy in such cases. In our prehospital system and in many European countries, trained physicians can be dispatched by EMCCs to provide medical care in the field [7], and can therefore draw and analyze ABG. However, even though recent and compact point-of-care testing devices allow for quick and reliable ABG analysis in the field [8], most emergency medical systems still lack such tools in their standard equipment. Barriers to the implementation of ABG analysis devices in the field have scarcely been described, but might include the absence of prehospital emergency physicians to interpret the results and the fear of unnecessarily delaying transport. Prehospital systems unable to dispatch emergency physicians in the field could however develop specific procedures to allow the use of and interpretation of ABG. Paramedics could be trained to draw arterial blood, protocols could be created to help paramedics determine cases in which ABG analysis could be of use, and distance interpretation by medical supervisors could be considered. Similar procedures have been successfully developed regarding electrocardiographic interpretation [9,10]. In the absence of distance medical supervision, regular algorithms or even electronic apps could be developed to facilitate the interpretation of ABG results [11,12]. Unnecessarily delaying transport could be avoided by limiting and clearly identifying the indications to ABG analysis, and by limiting the number of attempts and the time allowed to draw arterial blood. Moreover, other procedures can be performed while blood is drawn and analyzed, thereby further limiting potential delays. In our system, physicians are allowed only one attempt in a maximum of 60 s. Absolute contraindications to ABG sampling can usually be clinically identified easily (local infection, thrombosis or distorted anatomy, severe peripheral vascular disease, abnormal Allen’s test, etc.), and serious complications (infection, arterial occlusion or embolism, nerve injury, pseudo-aneurysm or vessel laceration, etc.) are rare. Minor complications (local pain, minor bleeding or hematoma, vasovagal response, etc.) can be easily managed and anticoagulants or antiplatelet agents do not represent absolute contraindications but often require only increased vascular pressure. Although venous sampling could lead to misdiagnosis, providers can be trained to recognize such samples. In this particular case, onsite ABG analysis allowed the physician to suspect metformin-associated metabolic acidosis and start the appropriate treatment even in the pre-hospital setting. While previous studies have failed to show a general positive effect of prehospital ABG on diagnostic accuracy, it can be of significant benefit in specific situations [13]. Moreover, ABG has been shown to improve treatment quality in critically ill patients [13]. In our system, prehospital physicians notify the Emergency Department (ED) team through the EMCC and can activate specific medical support before arrival. Consultants are thus notified before the patient’s arrival in the ED, thereby accelerating the initiation of appropriate and definitive care. In our institution, the decision to initiate haemodialysis is made either by nephrologists or by ICU physicians. In this particular case, the prehospital physician asked for the ICU consultant to be present upon arrival in the ED based on the suspected diagnosis. The patient was therefore quickly assessed by a multidisciplinary team, and therapeutic decisions were rapidly made. All further necessary steps (intubation, ICU admission, dialysis catheter insertion, haemodialysis initiation) were quickly initiated, and the time spent in the ED was therefore limited to a minimum. Metformin-associated metabolic acidosis is a rare but well-known complication of metformin treatment. Its incidence was calculated to be 4.3 cases per 100,000 patient-years [14]. When it develops, mortality rates as high as 48% have been reported [15,16]. Clinically significant metformin intoxication and lactic acid accumulation almost always occur in the presence of comorbid conditions, such as impaired kidney function. In our case, the patient indeed presented a KDIGO stage 3 acute kidney injury due to pre-renal disease consecutive to severe dehydration and sepsis. Prompt identification and treatment are mandatory in such cases, but diagnosis might be particularly difficult because of aspecific presentation. Moreover, acidaemia can be profound even though the patient does not seem to be critically ill. Although the main cause of the lactic acidosis found on ABG analysis was rapidly identified in this case, other potential aetiologies should be always considered. These include severe sepsis, shock, anemia, seizures, hypoxemia, carbon monoxide or cyanide poisoning, ethylene glycol or salicylates intoxication, alcoholism and liver disease, beta-agonist medication or profound thiamine deficit [17]. In our situation, these aetiologies were rapidly excluded by clinical appreciation and laboratory analysis. The role of bicarbonates infusion is still debated in metformin-associated metabolic acidosis as in other causes of anion gap metabolic acidosis [18]. In our case, this treatment was administered given the profound acidaemia and the signs of respiratory exhaustion, with the aim of avoiding endotracheal intubation. As the patient did not improve under supportive care and bicarbonate infusion, the decision to employ extracorporeal removal was swiftly made and, by virtue of a rapid diagnostic path, haemodialysis was started approximately 6 hours after the first medical contact. Haemodialysis has been shown to be effective in cases of metformin-associated metabolic acidoses [19] and is recommended in patients showing signs of severe poisoning. These signs include a lactate concentration higher than 20 mmol/L, a pH ≤ 7.0, or failure to improve with standard treatment within a few hours [4] as in our case. 4. Conclusions This case report shows that prehospital ABG measurement is feasible and can improve diagnostic accuracy in the field. In this particular case, ABG analysis enabled the prehospital physician to make the correct diagnosis in a timely manner, thus allowing the appropriate treatment to be initiated before hospital admission. Unnecessary delay and potential harm to the patient were thereby avoided. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author Contributions Writing—original draft preparation, P.E.F. and L.S.; writing—review and editing, P.E.F. and L.S. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Not applicable. Informed Consent Statement Informed consent was obtained from the patient whose case is reported in this manuscript. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest.	METFORMIN HYDROCHLORIDE	DrugsGivenReaction	CC BY	33466796	18,853,427	2021-01-14
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Dehydration'.	Prehospital Diagnosis of Shortness of Breath Caused by Profound Metformin-Associated Metabolic Acidosis. Shortness of breath is a common complaint among patients in emergency medicine. While most common causes are usually promptly identified, less frequent aetiologies might be challenging to diagnose, especially in the pre-hospital setting. We report a case of prehospital dyspnoea initially ascribed to pulmonary oedema which turned out to be the result of profound metformin-associated metabolic acidosis. This diagnosis was already made during the prehospital phase by virtue of arterial blood gas measurement. Pre-hospital measurement of arterial blood gases is therefore feasible and can improve diagnostic accuracy in the field, thus avoiding unnecessary delay and potential harm to the patient before initiating the appropriate therapeutic actions. 1. Introduction Shortness of breath (SOB) is one of the most common complaints among patients in prehospital emergency medicine [1]. Most common causes of shortness of breath (pulmonary oedema, pneumonia, chronic obstructive pulmonary disease exacerbation, pulmonary embolism, pneumothorax, etc.) are usually identified rather easily, although less common aetiologies might be overlooked and therefore misdiagnosed if clinicians fail to consider them. Furthermore, even a high clinical suspicion does not equal a definitive diagnosis, which might require para-clinical confirmation through specific investigations such as arterial blood gas (ABG) analysis. We report the case of a patient complaining of SOB that was initially imputed to pulmonary oedema. However, an ABG analysis performed in the field allowed the prehospital emergency physician to identify a profound metformin-associated metabolic acidosis as the actual cause of the patient’s dyspnoea. 2. Case Description The emergency medical call centre (EMCC) dispatched an advanced life support ambulance to take care of a 71-year-old woman who had called complaining about SOB. Upon arrival, prehospital providers noted shallow breathing with a respiratory rate of over 35 breaths per minute. Lung fields were clear to auscultation and the patient showed no sign of cyanosis. There was no haemodynamic compromise. The Glasgow Coma Scale was 15 with no focal deficit and the patient was normothermic. Given the respiratory distress, paramedics initiated oxygen therapy using a high-flow non-rebreather mask. Suspecting acute heart failure (AHF), they asked the EMCC to dispatch a medical mobile unit called ‘Service Mobile d’Urgence et de Réanimation’ (SMUR). These units are staffed by an advanced paramedic and a physician and can be dispatched to assist an ambulance in case of life-threatening emergencies [2]. These SMURs are the only prehospital units equipped with non-invasive ventilation (NIV) devices and portable blood gas analysers (iStat, Abbott Point of Care Inc., Princeton, NJ, USA) and are therefore often called upon to enhance the management of patients in acute respiratory distress or failure. While awaiting the arrival of the SMUR, the paramedics continued their evaluation and discovered that the patient was under metformin for diabetes and diuretics for hypertension, and that she was on day 3 of antibiotic treatment for urinary tract infection. She did not use cigarettes, alcohol, or illicit drugs. The patient was found to be hypoglycaemic at 2.3 mmol/L and received intravenous glucose accordingly. An ABG analysis performed by the SMUR physician shortly after arriving on site showed severe lactic acidosis (FiO2 65%: pH 6.845, PaCO2 1.95 kPa, PaO2 27.7 kPa, HCO3−—2.5 mmol/L, lactate 17.0 mmol/L). While AHF was suspected at first, the profound metabolic acidosis revealed by the ABG analysis was not compatible with the clinical picture. Indeed, the patient was not in cardiogenic shock, and a diagnosis of lactic acidosis secondary to metformin intoxication was thereafter suspected. Intravenous hydration was started and bicarbonates were administered. Since the patient showed signs of respiratory distress and exhaustion, NIV was initiated. In-hospital investigations confirmed severe lactic acidosis and acute kidney injury KDIGO stage 3 [3], with a creatinine of 788 µmol/L which yielded an estimated glomerular filtration rate of 5 mL/min/m2 according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. This kidney injury was secondary to the pre-renal disease on severe dehydration (diuretics, decreased drinking inputs, etc.) and sepsis, with consequent acute tubular necrosis. The patient showed no sign of hepatic impairment nor hypoxaemia. There was no hint that the patient’s illness could have been secondary to suicidal or accidental biguanide ingestion. The diagnosis of profound metformin-associated metabolic acidosis secondary to renal failure was definitively established at this stage. Despite aggressive hydration and bicarbonates infusion, the patient remained oligo-anuric with severe metabolic acidosis and showed signs of respiratory exhaustion finally requiring endotracheal intubation. She was admitted to the intensive care unit (ICU) for haemodynamic and respiratory management. The decision to start haemodialysis was finally made basing on the severity of acidosis and lactate level, coexisting renal failure and failure to improve with bicarbonate therapy [4]. The patient improved rapidly under this treatment and was extubated on the next day after pH normalisation and lactate clearance. She was transferred to the internal medicine ward after 48 h and finally discharged from the hospital 10 days later, after full renal recovery. 3. Discussion SOB is a major complaint among pre-hospital and ED patients, accounting for over 3.9 million visits in 2017 in the United States [5]. Most of these visits involve patients over 65 years old. Generally, cases of SOB are readily identified and can be ascribed to frequent aetiologies, i.e., heart failure (43%), community-acquired pneumonia (35%), acute exacerbation of chronic respiratory disease (32%), pulmonary embolism (18%), and acute asthma (3%). Two coexisting causes of SOB are found in as many as 47% of patients [6]. Nevertheless, clinicians must be aware that other less frequent aetiologies of SOB should be considered (salicylate intoxication, carbon monoxide poisoning, toxin-related metabolic acidosis, diabetic ketoacidosis, sepsis, anaemia, etc.). They should also acknowledge that diagnosing infrequent causes of SOB might be challenging, especially in the pre-hospital setting, where resources are limited and most investigation tools unavailable. Inappropriate dismissal of infrequent causes of SOB might lead to delayed diagnosis and treatment. Such delays might prove harmful, most particularly in old and frail patients. Our case exemplifies how pre-hospital ABG measurement can easily improve diagnostic accuracy in such cases. In our prehospital system and in many European countries, trained physicians can be dispatched by EMCCs to provide medical care in the field [7], and can therefore draw and analyze ABG. However, even though recent and compact point-of-care testing devices allow for quick and reliable ABG analysis in the field [8], most emergency medical systems still lack such tools in their standard equipment. Barriers to the implementation of ABG analysis devices in the field have scarcely been described, but might include the absence of prehospital emergency physicians to interpret the results and the fear of unnecessarily delaying transport. Prehospital systems unable to dispatch emergency physicians in the field could however develop specific procedures to allow the use of and interpretation of ABG. Paramedics could be trained to draw arterial blood, protocols could be created to help paramedics determine cases in which ABG analysis could be of use, and distance interpretation by medical supervisors could be considered. Similar procedures have been successfully developed regarding electrocardiographic interpretation [9,10]. In the absence of distance medical supervision, regular algorithms or even electronic apps could be developed to facilitate the interpretation of ABG results [11,12]. Unnecessarily delaying transport could be avoided by limiting and clearly identifying the indications to ABG analysis, and by limiting the number of attempts and the time allowed to draw arterial blood. Moreover, other procedures can be performed while blood is drawn and analyzed, thereby further limiting potential delays. In our system, physicians are allowed only one attempt in a maximum of 60 s. Absolute contraindications to ABG sampling can usually be clinically identified easily (local infection, thrombosis or distorted anatomy, severe peripheral vascular disease, abnormal Allen’s test, etc.), and serious complications (infection, arterial occlusion or embolism, nerve injury, pseudo-aneurysm or vessel laceration, etc.) are rare. Minor complications (local pain, minor bleeding or hematoma, vasovagal response, etc.) can be easily managed and anticoagulants or antiplatelet agents do not represent absolute contraindications but often require only increased vascular pressure. Although venous sampling could lead to misdiagnosis, providers can be trained to recognize such samples. In this particular case, onsite ABG analysis allowed the physician to suspect metformin-associated metabolic acidosis and start the appropriate treatment even in the pre-hospital setting. While previous studies have failed to show a general positive effect of prehospital ABG on diagnostic accuracy, it can be of significant benefit in specific situations [13]. Moreover, ABG has been shown to improve treatment quality in critically ill patients [13]. In our system, prehospital physicians notify the Emergency Department (ED) team through the EMCC and can activate specific medical support before arrival. Consultants are thus notified before the patient’s arrival in the ED, thereby accelerating the initiation of appropriate and definitive care. In our institution, the decision to initiate haemodialysis is made either by nephrologists or by ICU physicians. In this particular case, the prehospital physician asked for the ICU consultant to be present upon arrival in the ED based on the suspected diagnosis. The patient was therefore quickly assessed by a multidisciplinary team, and therapeutic decisions were rapidly made. All further necessary steps (intubation, ICU admission, dialysis catheter insertion, haemodialysis initiation) were quickly initiated, and the time spent in the ED was therefore limited to a minimum. Metformin-associated metabolic acidosis is a rare but well-known complication of metformin treatment. Its incidence was calculated to be 4.3 cases per 100,000 patient-years [14]. When it develops, mortality rates as high as 48% have been reported [15,16]. Clinically significant metformin intoxication and lactic acid accumulation almost always occur in the presence of comorbid conditions, such as impaired kidney function. In our case, the patient indeed presented a KDIGO stage 3 acute kidney injury due to pre-renal disease consecutive to severe dehydration and sepsis. Prompt identification and treatment are mandatory in such cases, but diagnosis might be particularly difficult because of aspecific presentation. Moreover, acidaemia can be profound even though the patient does not seem to be critically ill. Although the main cause of the lactic acidosis found on ABG analysis was rapidly identified in this case, other potential aetiologies should be always considered. These include severe sepsis, shock, anemia, seizures, hypoxemia, carbon monoxide or cyanide poisoning, ethylene glycol or salicylates intoxication, alcoholism and liver disease, beta-agonist medication or profound thiamine deficit [17]. In our situation, these aetiologies were rapidly excluded by clinical appreciation and laboratory analysis. The role of bicarbonates infusion is still debated in metformin-associated metabolic acidosis as in other causes of anion gap metabolic acidosis [18]. In our case, this treatment was administered given the profound acidaemia and the signs of respiratory exhaustion, with the aim of avoiding endotracheal intubation. As the patient did not improve under supportive care and bicarbonate infusion, the decision to employ extracorporeal removal was swiftly made and, by virtue of a rapid diagnostic path, haemodialysis was started approximately 6 hours after the first medical contact. Haemodialysis has been shown to be effective in cases of metformin-associated metabolic acidoses [19] and is recommended in patients showing signs of severe poisoning. These signs include a lactate concentration higher than 20 mmol/L, a pH ≤ 7.0, or failure to improve with standard treatment within a few hours [4] as in our case. 4. Conclusions This case report shows that prehospital ABG measurement is feasible and can improve diagnostic accuracy in the field. In this particular case, ABG analysis enabled the prehospital physician to make the correct diagnosis in a timely manner, thus allowing the appropriate treatment to be initiated before hospital admission. Unnecessary delay and potential harm to the patient were thereby avoided. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author Contributions Writing—original draft preparation, P.E.F. and L.S.; writing—review and editing, P.E.F. and L.S. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Not applicable. Informed Consent Statement Informed consent was obtained from the patient whose case is reported in this manuscript. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest.	METFORMIN HYDROCHLORIDE	DrugsGivenReaction	CC BY	33466796	18,853,427	2021-01-14
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Dyspnoea'.	Prehospital Diagnosis of Shortness of Breath Caused by Profound Metformin-Associated Metabolic Acidosis. Shortness of breath is a common complaint among patients in emergency medicine. While most common causes are usually promptly identified, less frequent aetiologies might be challenging to diagnose, especially in the pre-hospital setting. We report a case of prehospital dyspnoea initially ascribed to pulmonary oedema which turned out to be the result of profound metformin-associated metabolic acidosis. This diagnosis was already made during the prehospital phase by virtue of arterial blood gas measurement. Pre-hospital measurement of arterial blood gases is therefore feasible and can improve diagnostic accuracy in the field, thus avoiding unnecessary delay and potential harm to the patient before initiating the appropriate therapeutic actions. 1. Introduction Shortness of breath (SOB) is one of the most common complaints among patients in prehospital emergency medicine [1]. Most common causes of shortness of breath (pulmonary oedema, pneumonia, chronic obstructive pulmonary disease exacerbation, pulmonary embolism, pneumothorax, etc.) are usually identified rather easily, although less common aetiologies might be overlooked and therefore misdiagnosed if clinicians fail to consider them. Furthermore, even a high clinical suspicion does not equal a definitive diagnosis, which might require para-clinical confirmation through specific investigations such as arterial blood gas (ABG) analysis. We report the case of a patient complaining of SOB that was initially imputed to pulmonary oedema. However, an ABG analysis performed in the field allowed the prehospital emergency physician to identify a profound metformin-associated metabolic acidosis as the actual cause of the patient’s dyspnoea. 2. Case Description The emergency medical call centre (EMCC) dispatched an advanced life support ambulance to take care of a 71-year-old woman who had called complaining about SOB. Upon arrival, prehospital providers noted shallow breathing with a respiratory rate of over 35 breaths per minute. Lung fields were clear to auscultation and the patient showed no sign of cyanosis. There was no haemodynamic compromise. The Glasgow Coma Scale was 15 with no focal deficit and the patient was normothermic. Given the respiratory distress, paramedics initiated oxygen therapy using a high-flow non-rebreather mask. Suspecting acute heart failure (AHF), they asked the EMCC to dispatch a medical mobile unit called ‘Service Mobile d’Urgence et de Réanimation’ (SMUR). These units are staffed by an advanced paramedic and a physician and can be dispatched to assist an ambulance in case of life-threatening emergencies [2]. These SMURs are the only prehospital units equipped with non-invasive ventilation (NIV) devices and portable blood gas analysers (iStat, Abbott Point of Care Inc., Princeton, NJ, USA) and are therefore often called upon to enhance the management of patients in acute respiratory distress or failure. While awaiting the arrival of the SMUR, the paramedics continued their evaluation and discovered that the patient was under metformin for diabetes and diuretics for hypertension, and that she was on day 3 of antibiotic treatment for urinary tract infection. She did not use cigarettes, alcohol, or illicit drugs. The patient was found to be hypoglycaemic at 2.3 mmol/L and received intravenous glucose accordingly. An ABG analysis performed by the SMUR physician shortly after arriving on site showed severe lactic acidosis (FiO2 65%: pH 6.845, PaCO2 1.95 kPa, PaO2 27.7 kPa, HCO3−—2.5 mmol/L, lactate 17.0 mmol/L). While AHF was suspected at first, the profound metabolic acidosis revealed by the ABG analysis was not compatible with the clinical picture. Indeed, the patient was not in cardiogenic shock, and a diagnosis of lactic acidosis secondary to metformin intoxication was thereafter suspected. Intravenous hydration was started and bicarbonates were administered. Since the patient showed signs of respiratory distress and exhaustion, NIV was initiated. In-hospital investigations confirmed severe lactic acidosis and acute kidney injury KDIGO stage 3 [3], with a creatinine of 788 µmol/L which yielded an estimated glomerular filtration rate of 5 mL/min/m2 according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. This kidney injury was secondary to the pre-renal disease on severe dehydration (diuretics, decreased drinking inputs, etc.) and sepsis, with consequent acute tubular necrosis. The patient showed no sign of hepatic impairment nor hypoxaemia. There was no hint that the patient’s illness could have been secondary to suicidal or accidental biguanide ingestion. The diagnosis of profound metformin-associated metabolic acidosis secondary to renal failure was definitively established at this stage. Despite aggressive hydration and bicarbonates infusion, the patient remained oligo-anuric with severe metabolic acidosis and showed signs of respiratory exhaustion finally requiring endotracheal intubation. She was admitted to the intensive care unit (ICU) for haemodynamic and respiratory management. The decision to start haemodialysis was finally made basing on the severity of acidosis and lactate level, coexisting renal failure and failure to improve with bicarbonate therapy [4]. The patient improved rapidly under this treatment and was extubated on the next day after pH normalisation and lactate clearance. She was transferred to the internal medicine ward after 48 h and finally discharged from the hospital 10 days later, after full renal recovery. 3. Discussion SOB is a major complaint among pre-hospital and ED patients, accounting for over 3.9 million visits in 2017 in the United States [5]. Most of these visits involve patients over 65 years old. Generally, cases of SOB are readily identified and can be ascribed to frequent aetiologies, i.e., heart failure (43%), community-acquired pneumonia (35%), acute exacerbation of chronic respiratory disease (32%), pulmonary embolism (18%), and acute asthma (3%). Two coexisting causes of SOB are found in as many as 47% of patients [6]. Nevertheless, clinicians must be aware that other less frequent aetiologies of SOB should be considered (salicylate intoxication, carbon monoxide poisoning, toxin-related metabolic acidosis, diabetic ketoacidosis, sepsis, anaemia, etc.). They should also acknowledge that diagnosing infrequent causes of SOB might be challenging, especially in the pre-hospital setting, where resources are limited and most investigation tools unavailable. Inappropriate dismissal of infrequent causes of SOB might lead to delayed diagnosis and treatment. Such delays might prove harmful, most particularly in old and frail patients. Our case exemplifies how pre-hospital ABG measurement can easily improve diagnostic accuracy in such cases. In our prehospital system and in many European countries, trained physicians can be dispatched by EMCCs to provide medical care in the field [7], and can therefore draw and analyze ABG. However, even though recent and compact point-of-care testing devices allow for quick and reliable ABG analysis in the field [8], most emergency medical systems still lack such tools in their standard equipment. Barriers to the implementation of ABG analysis devices in the field have scarcely been described, but might include the absence of prehospital emergency physicians to interpret the results and the fear of unnecessarily delaying transport. Prehospital systems unable to dispatch emergency physicians in the field could however develop specific procedures to allow the use of and interpretation of ABG. Paramedics could be trained to draw arterial blood, protocols could be created to help paramedics determine cases in which ABG analysis could be of use, and distance interpretation by medical supervisors could be considered. Similar procedures have been successfully developed regarding electrocardiographic interpretation [9,10]. In the absence of distance medical supervision, regular algorithms or even electronic apps could be developed to facilitate the interpretation of ABG results [11,12]. Unnecessarily delaying transport could be avoided by limiting and clearly identifying the indications to ABG analysis, and by limiting the number of attempts and the time allowed to draw arterial blood. Moreover, other procedures can be performed while blood is drawn and analyzed, thereby further limiting potential delays. In our system, physicians are allowed only one attempt in a maximum of 60 s. Absolute contraindications to ABG sampling can usually be clinically identified easily (local infection, thrombosis or distorted anatomy, severe peripheral vascular disease, abnormal Allen’s test, etc.), and serious complications (infection, arterial occlusion or embolism, nerve injury, pseudo-aneurysm or vessel laceration, etc.) are rare. Minor complications (local pain, minor bleeding or hematoma, vasovagal response, etc.) can be easily managed and anticoagulants or antiplatelet agents do not represent absolute contraindications but often require only increased vascular pressure. Although venous sampling could lead to misdiagnosis, providers can be trained to recognize such samples. In this particular case, onsite ABG analysis allowed the physician to suspect metformin-associated metabolic acidosis and start the appropriate treatment even in the pre-hospital setting. While previous studies have failed to show a general positive effect of prehospital ABG on diagnostic accuracy, it can be of significant benefit in specific situations [13]. Moreover, ABG has been shown to improve treatment quality in critically ill patients [13]. In our system, prehospital physicians notify the Emergency Department (ED) team through the EMCC and can activate specific medical support before arrival. Consultants are thus notified before the patient’s arrival in the ED, thereby accelerating the initiation of appropriate and definitive care. In our institution, the decision to initiate haemodialysis is made either by nephrologists or by ICU physicians. In this particular case, the prehospital physician asked for the ICU consultant to be present upon arrival in the ED based on the suspected diagnosis. The patient was therefore quickly assessed by a multidisciplinary team, and therapeutic decisions were rapidly made. All further necessary steps (intubation, ICU admission, dialysis catheter insertion, haemodialysis initiation) were quickly initiated, and the time spent in the ED was therefore limited to a minimum. Metformin-associated metabolic acidosis is a rare but well-known complication of metformin treatment. Its incidence was calculated to be 4.3 cases per 100,000 patient-years [14]. When it develops, mortality rates as high as 48% have been reported [15,16]. Clinically significant metformin intoxication and lactic acid accumulation almost always occur in the presence of comorbid conditions, such as impaired kidney function. In our case, the patient indeed presented a KDIGO stage 3 acute kidney injury due to pre-renal disease consecutive to severe dehydration and sepsis. Prompt identification and treatment are mandatory in such cases, but diagnosis might be particularly difficult because of aspecific presentation. Moreover, acidaemia can be profound even though the patient does not seem to be critically ill. Although the main cause of the lactic acidosis found on ABG analysis was rapidly identified in this case, other potential aetiologies should be always considered. These include severe sepsis, shock, anemia, seizures, hypoxemia, carbon monoxide or cyanide poisoning, ethylene glycol or salicylates intoxication, alcoholism and liver disease, beta-agonist medication or profound thiamine deficit [17]. In our situation, these aetiologies were rapidly excluded by clinical appreciation and laboratory analysis. The role of bicarbonates infusion is still debated in metformin-associated metabolic acidosis as in other causes of anion gap metabolic acidosis [18]. In our case, this treatment was administered given the profound acidaemia and the signs of respiratory exhaustion, with the aim of avoiding endotracheal intubation. As the patient did not improve under supportive care and bicarbonate infusion, the decision to employ extracorporeal removal was swiftly made and, by virtue of a rapid diagnostic path, haemodialysis was started approximately 6 hours after the first medical contact. Haemodialysis has been shown to be effective in cases of metformin-associated metabolic acidoses [19] and is recommended in patients showing signs of severe poisoning. These signs include a lactate concentration higher than 20 mmol/L, a pH ≤ 7.0, or failure to improve with standard treatment within a few hours [4] as in our case. 4. Conclusions This case report shows that prehospital ABG measurement is feasible and can improve diagnostic accuracy in the field. In this particular case, ABG analysis enabled the prehospital physician to make the correct diagnosis in a timely manner, thus allowing the appropriate treatment to be initiated before hospital admission. Unnecessary delay and potential harm to the patient were thereby avoided. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author Contributions Writing—original draft preparation, P.E.F. and L.S.; writing—review and editing, P.E.F. and L.S. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Not applicable. Informed Consent Statement Informed consent was obtained from the patient whose case is reported in this manuscript. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest.	METFORMIN HYDROCHLORIDE	DrugsGivenReaction	CC BY	33466796	18,855,942	2021-01-14
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Metabolic acidosis'.	Prehospital Diagnosis of Shortness of Breath Caused by Profound Metformin-Associated Metabolic Acidosis. Shortness of breath is a common complaint among patients in emergency medicine. While most common causes are usually promptly identified, less frequent aetiologies might be challenging to diagnose, especially in the pre-hospital setting. We report a case of prehospital dyspnoea initially ascribed to pulmonary oedema which turned out to be the result of profound metformin-associated metabolic acidosis. This diagnosis was already made during the prehospital phase by virtue of arterial blood gas measurement. Pre-hospital measurement of arterial blood gases is therefore feasible and can improve diagnostic accuracy in the field, thus avoiding unnecessary delay and potential harm to the patient before initiating the appropriate therapeutic actions. 1. Introduction Shortness of breath (SOB) is one of the most common complaints among patients in prehospital emergency medicine [1]. Most common causes of shortness of breath (pulmonary oedema, pneumonia, chronic obstructive pulmonary disease exacerbation, pulmonary embolism, pneumothorax, etc.) are usually identified rather easily, although less common aetiologies might be overlooked and therefore misdiagnosed if clinicians fail to consider them. Furthermore, even a high clinical suspicion does not equal a definitive diagnosis, which might require para-clinical confirmation through specific investigations such as arterial blood gas (ABG) analysis. We report the case of a patient complaining of SOB that was initially imputed to pulmonary oedema. However, an ABG analysis performed in the field allowed the prehospital emergency physician to identify a profound metformin-associated metabolic acidosis as the actual cause of the patient’s dyspnoea. 2. Case Description The emergency medical call centre (EMCC) dispatched an advanced life support ambulance to take care of a 71-year-old woman who had called complaining about SOB. Upon arrival, prehospital providers noted shallow breathing with a respiratory rate of over 35 breaths per minute. Lung fields were clear to auscultation and the patient showed no sign of cyanosis. There was no haemodynamic compromise. The Glasgow Coma Scale was 15 with no focal deficit and the patient was normothermic. Given the respiratory distress, paramedics initiated oxygen therapy using a high-flow non-rebreather mask. Suspecting acute heart failure (AHF), they asked the EMCC to dispatch a medical mobile unit called ‘Service Mobile d’Urgence et de Réanimation’ (SMUR). These units are staffed by an advanced paramedic and a physician and can be dispatched to assist an ambulance in case of life-threatening emergencies [2]. These SMURs are the only prehospital units equipped with non-invasive ventilation (NIV) devices and portable blood gas analysers (iStat, Abbott Point of Care Inc., Princeton, NJ, USA) and are therefore often called upon to enhance the management of patients in acute respiratory distress or failure. While awaiting the arrival of the SMUR, the paramedics continued their evaluation and discovered that the patient was under metformin for diabetes and diuretics for hypertension, and that she was on day 3 of antibiotic treatment for urinary tract infection. She did not use cigarettes, alcohol, or illicit drugs. The patient was found to be hypoglycaemic at 2.3 mmol/L and received intravenous glucose accordingly. An ABG analysis performed by the SMUR physician shortly after arriving on site showed severe lactic acidosis (FiO2 65%: pH 6.845, PaCO2 1.95 kPa, PaO2 27.7 kPa, HCO3−—2.5 mmol/L, lactate 17.0 mmol/L). While AHF was suspected at first, the profound metabolic acidosis revealed by the ABG analysis was not compatible with the clinical picture. Indeed, the patient was not in cardiogenic shock, and a diagnosis of lactic acidosis secondary to metformin intoxication was thereafter suspected. Intravenous hydration was started and bicarbonates were administered. Since the patient showed signs of respiratory distress and exhaustion, NIV was initiated. In-hospital investigations confirmed severe lactic acidosis and acute kidney injury KDIGO stage 3 [3], with a creatinine of 788 µmol/L which yielded an estimated glomerular filtration rate of 5 mL/min/m2 according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. This kidney injury was secondary to the pre-renal disease on severe dehydration (diuretics, decreased drinking inputs, etc.) and sepsis, with consequent acute tubular necrosis. The patient showed no sign of hepatic impairment nor hypoxaemia. There was no hint that the patient’s illness could have been secondary to suicidal or accidental biguanide ingestion. The diagnosis of profound metformin-associated metabolic acidosis secondary to renal failure was definitively established at this stage. Despite aggressive hydration and bicarbonates infusion, the patient remained oligo-anuric with severe metabolic acidosis and showed signs of respiratory exhaustion finally requiring endotracheal intubation. She was admitted to the intensive care unit (ICU) for haemodynamic and respiratory management. The decision to start haemodialysis was finally made basing on the severity of acidosis and lactate level, coexisting renal failure and failure to improve with bicarbonate therapy [4]. The patient improved rapidly under this treatment and was extubated on the next day after pH normalisation and lactate clearance. She was transferred to the internal medicine ward after 48 h and finally discharged from the hospital 10 days later, after full renal recovery. 3. Discussion SOB is a major complaint among pre-hospital and ED patients, accounting for over 3.9 million visits in 2017 in the United States [5]. Most of these visits involve patients over 65 years old. Generally, cases of SOB are readily identified and can be ascribed to frequent aetiologies, i.e., heart failure (43%), community-acquired pneumonia (35%), acute exacerbation of chronic respiratory disease (32%), pulmonary embolism (18%), and acute asthma (3%). Two coexisting causes of SOB are found in as many as 47% of patients [6]. Nevertheless, clinicians must be aware that other less frequent aetiologies of SOB should be considered (salicylate intoxication, carbon monoxide poisoning, toxin-related metabolic acidosis, diabetic ketoacidosis, sepsis, anaemia, etc.). They should also acknowledge that diagnosing infrequent causes of SOB might be challenging, especially in the pre-hospital setting, where resources are limited and most investigation tools unavailable. Inappropriate dismissal of infrequent causes of SOB might lead to delayed diagnosis and treatment. Such delays might prove harmful, most particularly in old and frail patients. Our case exemplifies how pre-hospital ABG measurement can easily improve diagnostic accuracy in such cases. In our prehospital system and in many European countries, trained physicians can be dispatched by EMCCs to provide medical care in the field [7], and can therefore draw and analyze ABG. However, even though recent and compact point-of-care testing devices allow for quick and reliable ABG analysis in the field [8], most emergency medical systems still lack such tools in their standard equipment. Barriers to the implementation of ABG analysis devices in the field have scarcely been described, but might include the absence of prehospital emergency physicians to interpret the results and the fear of unnecessarily delaying transport. Prehospital systems unable to dispatch emergency physicians in the field could however develop specific procedures to allow the use of and interpretation of ABG. Paramedics could be trained to draw arterial blood, protocols could be created to help paramedics determine cases in which ABG analysis could be of use, and distance interpretation by medical supervisors could be considered. Similar procedures have been successfully developed regarding electrocardiographic interpretation [9,10]. In the absence of distance medical supervision, regular algorithms or even electronic apps could be developed to facilitate the interpretation of ABG results [11,12]. Unnecessarily delaying transport could be avoided by limiting and clearly identifying the indications to ABG analysis, and by limiting the number of attempts and the time allowed to draw arterial blood. Moreover, other procedures can be performed while blood is drawn and analyzed, thereby further limiting potential delays. In our system, physicians are allowed only one attempt in a maximum of 60 s. Absolute contraindications to ABG sampling can usually be clinically identified easily (local infection, thrombosis or distorted anatomy, severe peripheral vascular disease, abnormal Allen’s test, etc.), and serious complications (infection, arterial occlusion or embolism, nerve injury, pseudo-aneurysm or vessel laceration, etc.) are rare. Minor complications (local pain, minor bleeding or hematoma, vasovagal response, etc.) can be easily managed and anticoagulants or antiplatelet agents do not represent absolute contraindications but often require only increased vascular pressure. Although venous sampling could lead to misdiagnosis, providers can be trained to recognize such samples. In this particular case, onsite ABG analysis allowed the physician to suspect metformin-associated metabolic acidosis and start the appropriate treatment even in the pre-hospital setting. While previous studies have failed to show a general positive effect of prehospital ABG on diagnostic accuracy, it can be of significant benefit in specific situations [13]. Moreover, ABG has been shown to improve treatment quality in critically ill patients [13]. In our system, prehospital physicians notify the Emergency Department (ED) team through the EMCC and can activate specific medical support before arrival. Consultants are thus notified before the patient’s arrival in the ED, thereby accelerating the initiation of appropriate and definitive care. In our institution, the decision to initiate haemodialysis is made either by nephrologists or by ICU physicians. In this particular case, the prehospital physician asked for the ICU consultant to be present upon arrival in the ED based on the suspected diagnosis. The patient was therefore quickly assessed by a multidisciplinary team, and therapeutic decisions were rapidly made. All further necessary steps (intubation, ICU admission, dialysis catheter insertion, haemodialysis initiation) were quickly initiated, and the time spent in the ED was therefore limited to a minimum. Metformin-associated metabolic acidosis is a rare but well-known complication of metformin treatment. Its incidence was calculated to be 4.3 cases per 100,000 patient-years [14]. When it develops, mortality rates as high as 48% have been reported [15,16]. Clinically significant metformin intoxication and lactic acid accumulation almost always occur in the presence of comorbid conditions, such as impaired kidney function. In our case, the patient indeed presented a KDIGO stage 3 acute kidney injury due to pre-renal disease consecutive to severe dehydration and sepsis. Prompt identification and treatment are mandatory in such cases, but diagnosis might be particularly difficult because of aspecific presentation. Moreover, acidaemia can be profound even though the patient does not seem to be critically ill. Although the main cause of the lactic acidosis found on ABG analysis was rapidly identified in this case, other potential aetiologies should be always considered. These include severe sepsis, shock, anemia, seizures, hypoxemia, carbon monoxide or cyanide poisoning, ethylene glycol or salicylates intoxication, alcoholism and liver disease, beta-agonist medication or profound thiamine deficit [17]. In our situation, these aetiologies were rapidly excluded by clinical appreciation and laboratory analysis. The role of bicarbonates infusion is still debated in metformin-associated metabolic acidosis as in other causes of anion gap metabolic acidosis [18]. In our case, this treatment was administered given the profound acidaemia and the signs of respiratory exhaustion, with the aim of avoiding endotracheal intubation. As the patient did not improve under supportive care and bicarbonate infusion, the decision to employ extracorporeal removal was swiftly made and, by virtue of a rapid diagnostic path, haemodialysis was started approximately 6 hours after the first medical contact. Haemodialysis has been shown to be effective in cases of metformin-associated metabolic acidoses [19] and is recommended in patients showing signs of severe poisoning. These signs include a lactate concentration higher than 20 mmol/L, a pH ≤ 7.0, or failure to improve with standard treatment within a few hours [4] as in our case. 4. Conclusions This case report shows that prehospital ABG measurement is feasible and can improve diagnostic accuracy in the field. In this particular case, ABG analysis enabled the prehospital physician to make the correct diagnosis in a timely manner, thus allowing the appropriate treatment to be initiated before hospital admission. Unnecessary delay and potential harm to the patient were thereby avoided. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author Contributions Writing—original draft preparation, P.E.F. and L.S.; writing—review and editing, P.E.F. and L.S. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Not applicable. Informed Consent Statement Informed consent was obtained from the patient whose case is reported in this manuscript. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest.	METFORMIN HYDROCHLORIDE	DrugsGivenReaction	CC BY	33466796	18,853,427	2021-01-14
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Renal tubular necrosis'.	Prehospital Diagnosis of Shortness of Breath Caused by Profound Metformin-Associated Metabolic Acidosis. Shortness of breath is a common complaint among patients in emergency medicine. While most common causes are usually promptly identified, less frequent aetiologies might be challenging to diagnose, especially in the pre-hospital setting. We report a case of prehospital dyspnoea initially ascribed to pulmonary oedema which turned out to be the result of profound metformin-associated metabolic acidosis. This diagnosis was already made during the prehospital phase by virtue of arterial blood gas measurement. Pre-hospital measurement of arterial blood gases is therefore feasible and can improve diagnostic accuracy in the field, thus avoiding unnecessary delay and potential harm to the patient before initiating the appropriate therapeutic actions. 1. Introduction Shortness of breath (SOB) is one of the most common complaints among patients in prehospital emergency medicine [1]. Most common causes of shortness of breath (pulmonary oedema, pneumonia, chronic obstructive pulmonary disease exacerbation, pulmonary embolism, pneumothorax, etc.) are usually identified rather easily, although less common aetiologies might be overlooked and therefore misdiagnosed if clinicians fail to consider them. Furthermore, even a high clinical suspicion does not equal a definitive diagnosis, which might require para-clinical confirmation through specific investigations such as arterial blood gas (ABG) analysis. We report the case of a patient complaining of SOB that was initially imputed to pulmonary oedema. However, an ABG analysis performed in the field allowed the prehospital emergency physician to identify a profound metformin-associated metabolic acidosis as the actual cause of the patient’s dyspnoea. 2. Case Description The emergency medical call centre (EMCC) dispatched an advanced life support ambulance to take care of a 71-year-old woman who had called complaining about SOB. Upon arrival, prehospital providers noted shallow breathing with a respiratory rate of over 35 breaths per minute. Lung fields were clear to auscultation and the patient showed no sign of cyanosis. There was no haemodynamic compromise. The Glasgow Coma Scale was 15 with no focal deficit and the patient was normothermic. Given the respiratory distress, paramedics initiated oxygen therapy using a high-flow non-rebreather mask. Suspecting acute heart failure (AHF), they asked the EMCC to dispatch a medical mobile unit called ‘Service Mobile d’Urgence et de Réanimation’ (SMUR). These units are staffed by an advanced paramedic and a physician and can be dispatched to assist an ambulance in case of life-threatening emergencies [2]. These SMURs are the only prehospital units equipped with non-invasive ventilation (NIV) devices and portable blood gas analysers (iStat, Abbott Point of Care Inc., Princeton, NJ, USA) and are therefore often called upon to enhance the management of patients in acute respiratory distress or failure. While awaiting the arrival of the SMUR, the paramedics continued their evaluation and discovered that the patient was under metformin for diabetes and diuretics for hypertension, and that she was on day 3 of antibiotic treatment for urinary tract infection. She did not use cigarettes, alcohol, or illicit drugs. The patient was found to be hypoglycaemic at 2.3 mmol/L and received intravenous glucose accordingly. An ABG analysis performed by the SMUR physician shortly after arriving on site showed severe lactic acidosis (FiO2 65%: pH 6.845, PaCO2 1.95 kPa, PaO2 27.7 kPa, HCO3−—2.5 mmol/L, lactate 17.0 mmol/L). While AHF was suspected at first, the profound metabolic acidosis revealed by the ABG analysis was not compatible with the clinical picture. Indeed, the patient was not in cardiogenic shock, and a diagnosis of lactic acidosis secondary to metformin intoxication was thereafter suspected. Intravenous hydration was started and bicarbonates were administered. Since the patient showed signs of respiratory distress and exhaustion, NIV was initiated. In-hospital investigations confirmed severe lactic acidosis and acute kidney injury KDIGO stage 3 [3], with a creatinine of 788 µmol/L which yielded an estimated glomerular filtration rate of 5 mL/min/m2 according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. This kidney injury was secondary to the pre-renal disease on severe dehydration (diuretics, decreased drinking inputs, etc.) and sepsis, with consequent acute tubular necrosis. The patient showed no sign of hepatic impairment nor hypoxaemia. There was no hint that the patient’s illness could have been secondary to suicidal or accidental biguanide ingestion. The diagnosis of profound metformin-associated metabolic acidosis secondary to renal failure was definitively established at this stage. Despite aggressive hydration and bicarbonates infusion, the patient remained oligo-anuric with severe metabolic acidosis and showed signs of respiratory exhaustion finally requiring endotracheal intubation. She was admitted to the intensive care unit (ICU) for haemodynamic and respiratory management. The decision to start haemodialysis was finally made basing on the severity of acidosis and lactate level, coexisting renal failure and failure to improve with bicarbonate therapy [4]. The patient improved rapidly under this treatment and was extubated on the next day after pH normalisation and lactate clearance. She was transferred to the internal medicine ward after 48 h and finally discharged from the hospital 10 days later, after full renal recovery. 3. Discussion SOB is a major complaint among pre-hospital and ED patients, accounting for over 3.9 million visits in 2017 in the United States [5]. Most of these visits involve patients over 65 years old. Generally, cases of SOB are readily identified and can be ascribed to frequent aetiologies, i.e., heart failure (43%), community-acquired pneumonia (35%), acute exacerbation of chronic respiratory disease (32%), pulmonary embolism (18%), and acute asthma (3%). Two coexisting causes of SOB are found in as many as 47% of patients [6]. Nevertheless, clinicians must be aware that other less frequent aetiologies of SOB should be considered (salicylate intoxication, carbon monoxide poisoning, toxin-related metabolic acidosis, diabetic ketoacidosis, sepsis, anaemia, etc.). They should also acknowledge that diagnosing infrequent causes of SOB might be challenging, especially in the pre-hospital setting, where resources are limited and most investigation tools unavailable. Inappropriate dismissal of infrequent causes of SOB might lead to delayed diagnosis and treatment. Such delays might prove harmful, most particularly in old and frail patients. Our case exemplifies how pre-hospital ABG measurement can easily improve diagnostic accuracy in such cases. In our prehospital system and in many European countries, trained physicians can be dispatched by EMCCs to provide medical care in the field [7], and can therefore draw and analyze ABG. However, even though recent and compact point-of-care testing devices allow for quick and reliable ABG analysis in the field [8], most emergency medical systems still lack such tools in their standard equipment. Barriers to the implementation of ABG analysis devices in the field have scarcely been described, but might include the absence of prehospital emergency physicians to interpret the results and the fear of unnecessarily delaying transport. Prehospital systems unable to dispatch emergency physicians in the field could however develop specific procedures to allow the use of and interpretation of ABG. Paramedics could be trained to draw arterial blood, protocols could be created to help paramedics determine cases in which ABG analysis could be of use, and distance interpretation by medical supervisors could be considered. Similar procedures have been successfully developed regarding electrocardiographic interpretation [9,10]. In the absence of distance medical supervision, regular algorithms or even electronic apps could be developed to facilitate the interpretation of ABG results [11,12]. Unnecessarily delaying transport could be avoided by limiting and clearly identifying the indications to ABG analysis, and by limiting the number of attempts and the time allowed to draw arterial blood. Moreover, other procedures can be performed while blood is drawn and analyzed, thereby further limiting potential delays. In our system, physicians are allowed only one attempt in a maximum of 60 s. Absolute contraindications to ABG sampling can usually be clinically identified easily (local infection, thrombosis or distorted anatomy, severe peripheral vascular disease, abnormal Allen’s test, etc.), and serious complications (infection, arterial occlusion or embolism, nerve injury, pseudo-aneurysm or vessel laceration, etc.) are rare. Minor complications (local pain, minor bleeding or hematoma, vasovagal response, etc.) can be easily managed and anticoagulants or antiplatelet agents do not represent absolute contraindications but often require only increased vascular pressure. Although venous sampling could lead to misdiagnosis, providers can be trained to recognize such samples. In this particular case, onsite ABG analysis allowed the physician to suspect metformin-associated metabolic acidosis and start the appropriate treatment even in the pre-hospital setting. While previous studies have failed to show a general positive effect of prehospital ABG on diagnostic accuracy, it can be of significant benefit in specific situations [13]. Moreover, ABG has been shown to improve treatment quality in critically ill patients [13]. In our system, prehospital physicians notify the Emergency Department (ED) team through the EMCC and can activate specific medical support before arrival. Consultants are thus notified before the patient’s arrival in the ED, thereby accelerating the initiation of appropriate and definitive care. In our institution, the decision to initiate haemodialysis is made either by nephrologists or by ICU physicians. In this particular case, the prehospital physician asked for the ICU consultant to be present upon arrival in the ED based on the suspected diagnosis. The patient was therefore quickly assessed by a multidisciplinary team, and therapeutic decisions were rapidly made. All further necessary steps (intubation, ICU admission, dialysis catheter insertion, haemodialysis initiation) were quickly initiated, and the time spent in the ED was therefore limited to a minimum. Metformin-associated metabolic acidosis is a rare but well-known complication of metformin treatment. Its incidence was calculated to be 4.3 cases per 100,000 patient-years [14]. When it develops, mortality rates as high as 48% have been reported [15,16]. Clinically significant metformin intoxication and lactic acid accumulation almost always occur in the presence of comorbid conditions, such as impaired kidney function. In our case, the patient indeed presented a KDIGO stage 3 acute kidney injury due to pre-renal disease consecutive to severe dehydration and sepsis. Prompt identification and treatment are mandatory in such cases, but diagnosis might be particularly difficult because of aspecific presentation. Moreover, acidaemia can be profound even though the patient does not seem to be critically ill. Although the main cause of the lactic acidosis found on ABG analysis was rapidly identified in this case, other potential aetiologies should be always considered. These include severe sepsis, shock, anemia, seizures, hypoxemia, carbon monoxide or cyanide poisoning, ethylene glycol or salicylates intoxication, alcoholism and liver disease, beta-agonist medication or profound thiamine deficit [17]. In our situation, these aetiologies were rapidly excluded by clinical appreciation and laboratory analysis. The role of bicarbonates infusion is still debated in metformin-associated metabolic acidosis as in other causes of anion gap metabolic acidosis [18]. In our case, this treatment was administered given the profound acidaemia and the signs of respiratory exhaustion, with the aim of avoiding endotracheal intubation. As the patient did not improve under supportive care and bicarbonate infusion, the decision to employ extracorporeal removal was swiftly made and, by virtue of a rapid diagnostic path, haemodialysis was started approximately 6 hours after the first medical contact. Haemodialysis has been shown to be effective in cases of metformin-associated metabolic acidoses [19] and is recommended in patients showing signs of severe poisoning. These signs include a lactate concentration higher than 20 mmol/L, a pH ≤ 7.0, or failure to improve with standard treatment within a few hours [4] as in our case. 4. Conclusions This case report shows that prehospital ABG measurement is feasible and can improve diagnostic accuracy in the field. In this particular case, ABG analysis enabled the prehospital physician to make the correct diagnosis in a timely manner, thus allowing the appropriate treatment to be initiated before hospital admission. Unnecessary delay and potential harm to the patient were thereby avoided. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author Contributions Writing—original draft preparation, P.E.F. and L.S.; writing—review and editing, P.E.F. and L.S. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Not applicable. Informed Consent Statement Informed consent was obtained from the patient whose case is reported in this manuscript. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest.	METFORMIN HYDROCHLORIDE	DrugsGivenReaction	CC BY	33466796	18,855,942	2021-01-14
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Sepsis'.	Prehospital Diagnosis of Shortness of Breath Caused by Profound Metformin-Associated Metabolic Acidosis. Shortness of breath is a common complaint among patients in emergency medicine. While most common causes are usually promptly identified, less frequent aetiologies might be challenging to diagnose, especially in the pre-hospital setting. We report a case of prehospital dyspnoea initially ascribed to pulmonary oedema which turned out to be the result of profound metformin-associated metabolic acidosis. This diagnosis was already made during the prehospital phase by virtue of arterial blood gas measurement. Pre-hospital measurement of arterial blood gases is therefore feasible and can improve diagnostic accuracy in the field, thus avoiding unnecessary delay and potential harm to the patient before initiating the appropriate therapeutic actions. 1. Introduction Shortness of breath (SOB) is one of the most common complaints among patients in prehospital emergency medicine [1]. Most common causes of shortness of breath (pulmonary oedema, pneumonia, chronic obstructive pulmonary disease exacerbation, pulmonary embolism, pneumothorax, etc.) are usually identified rather easily, although less common aetiologies might be overlooked and therefore misdiagnosed if clinicians fail to consider them. Furthermore, even a high clinical suspicion does not equal a definitive diagnosis, which might require para-clinical confirmation through specific investigations such as arterial blood gas (ABG) analysis. We report the case of a patient complaining of SOB that was initially imputed to pulmonary oedema. However, an ABG analysis performed in the field allowed the prehospital emergency physician to identify a profound metformin-associated metabolic acidosis as the actual cause of the patient’s dyspnoea. 2. Case Description The emergency medical call centre (EMCC) dispatched an advanced life support ambulance to take care of a 71-year-old woman who had called complaining about SOB. Upon arrival, prehospital providers noted shallow breathing with a respiratory rate of over 35 breaths per minute. Lung fields were clear to auscultation and the patient showed no sign of cyanosis. There was no haemodynamic compromise. The Glasgow Coma Scale was 15 with no focal deficit and the patient was normothermic. Given the respiratory distress, paramedics initiated oxygen therapy using a high-flow non-rebreather mask. Suspecting acute heart failure (AHF), they asked the EMCC to dispatch a medical mobile unit called ‘Service Mobile d’Urgence et de Réanimation’ (SMUR). These units are staffed by an advanced paramedic and a physician and can be dispatched to assist an ambulance in case of life-threatening emergencies [2]. These SMURs are the only prehospital units equipped with non-invasive ventilation (NIV) devices and portable blood gas analysers (iStat, Abbott Point of Care Inc., Princeton, NJ, USA) and are therefore often called upon to enhance the management of patients in acute respiratory distress or failure. While awaiting the arrival of the SMUR, the paramedics continued their evaluation and discovered that the patient was under metformin for diabetes and diuretics for hypertension, and that she was on day 3 of antibiotic treatment for urinary tract infection. She did not use cigarettes, alcohol, or illicit drugs. The patient was found to be hypoglycaemic at 2.3 mmol/L and received intravenous glucose accordingly. An ABG analysis performed by the SMUR physician shortly after arriving on site showed severe lactic acidosis (FiO2 65%: pH 6.845, PaCO2 1.95 kPa, PaO2 27.7 kPa, HCO3−—2.5 mmol/L, lactate 17.0 mmol/L). While AHF was suspected at first, the profound metabolic acidosis revealed by the ABG analysis was not compatible with the clinical picture. Indeed, the patient was not in cardiogenic shock, and a diagnosis of lactic acidosis secondary to metformin intoxication was thereafter suspected. Intravenous hydration was started and bicarbonates were administered. Since the patient showed signs of respiratory distress and exhaustion, NIV was initiated. In-hospital investigations confirmed severe lactic acidosis and acute kidney injury KDIGO stage 3 [3], with a creatinine of 788 µmol/L which yielded an estimated glomerular filtration rate of 5 mL/min/m2 according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. This kidney injury was secondary to the pre-renal disease on severe dehydration (diuretics, decreased drinking inputs, etc.) and sepsis, with consequent acute tubular necrosis. The patient showed no sign of hepatic impairment nor hypoxaemia. There was no hint that the patient’s illness could have been secondary to suicidal or accidental biguanide ingestion. The diagnosis of profound metformin-associated metabolic acidosis secondary to renal failure was definitively established at this stage. Despite aggressive hydration and bicarbonates infusion, the patient remained oligo-anuric with severe metabolic acidosis and showed signs of respiratory exhaustion finally requiring endotracheal intubation. She was admitted to the intensive care unit (ICU) for haemodynamic and respiratory management. The decision to start haemodialysis was finally made basing on the severity of acidosis and lactate level, coexisting renal failure and failure to improve with bicarbonate therapy [4]. The patient improved rapidly under this treatment and was extubated on the next day after pH normalisation and lactate clearance. She was transferred to the internal medicine ward after 48 h and finally discharged from the hospital 10 days later, after full renal recovery. 3. Discussion SOB is a major complaint among pre-hospital and ED patients, accounting for over 3.9 million visits in 2017 in the United States [5]. Most of these visits involve patients over 65 years old. Generally, cases of SOB are readily identified and can be ascribed to frequent aetiologies, i.e., heart failure (43%), community-acquired pneumonia (35%), acute exacerbation of chronic respiratory disease (32%), pulmonary embolism (18%), and acute asthma (3%). Two coexisting causes of SOB are found in as many as 47% of patients [6]. Nevertheless, clinicians must be aware that other less frequent aetiologies of SOB should be considered (salicylate intoxication, carbon monoxide poisoning, toxin-related metabolic acidosis, diabetic ketoacidosis, sepsis, anaemia, etc.). They should also acknowledge that diagnosing infrequent causes of SOB might be challenging, especially in the pre-hospital setting, where resources are limited and most investigation tools unavailable. Inappropriate dismissal of infrequent causes of SOB might lead to delayed diagnosis and treatment. Such delays might prove harmful, most particularly in old and frail patients. Our case exemplifies how pre-hospital ABG measurement can easily improve diagnostic accuracy in such cases. In our prehospital system and in many European countries, trained physicians can be dispatched by EMCCs to provide medical care in the field [7], and can therefore draw and analyze ABG. However, even though recent and compact point-of-care testing devices allow for quick and reliable ABG analysis in the field [8], most emergency medical systems still lack such tools in their standard equipment. Barriers to the implementation of ABG analysis devices in the field have scarcely been described, but might include the absence of prehospital emergency physicians to interpret the results and the fear of unnecessarily delaying transport. Prehospital systems unable to dispatch emergency physicians in the field could however develop specific procedures to allow the use of and interpretation of ABG. Paramedics could be trained to draw arterial blood, protocols could be created to help paramedics determine cases in which ABG analysis could be of use, and distance interpretation by medical supervisors could be considered. Similar procedures have been successfully developed regarding electrocardiographic interpretation [9,10]. In the absence of distance medical supervision, regular algorithms or even electronic apps could be developed to facilitate the interpretation of ABG results [11,12]. Unnecessarily delaying transport could be avoided by limiting and clearly identifying the indications to ABG analysis, and by limiting the number of attempts and the time allowed to draw arterial blood. Moreover, other procedures can be performed while blood is drawn and analyzed, thereby further limiting potential delays. In our system, physicians are allowed only one attempt in a maximum of 60 s. Absolute contraindications to ABG sampling can usually be clinically identified easily (local infection, thrombosis or distorted anatomy, severe peripheral vascular disease, abnormal Allen’s test, etc.), and serious complications (infection, arterial occlusion or embolism, nerve injury, pseudo-aneurysm or vessel laceration, etc.) are rare. Minor complications (local pain, minor bleeding or hematoma, vasovagal response, etc.) can be easily managed and anticoagulants or antiplatelet agents do not represent absolute contraindications but often require only increased vascular pressure. Although venous sampling could lead to misdiagnosis, providers can be trained to recognize such samples. In this particular case, onsite ABG analysis allowed the physician to suspect metformin-associated metabolic acidosis and start the appropriate treatment even in the pre-hospital setting. While previous studies have failed to show a general positive effect of prehospital ABG on diagnostic accuracy, it can be of significant benefit in specific situations [13]. Moreover, ABG has been shown to improve treatment quality in critically ill patients [13]. In our system, prehospital physicians notify the Emergency Department (ED) team through the EMCC and can activate specific medical support before arrival. Consultants are thus notified before the patient’s arrival in the ED, thereby accelerating the initiation of appropriate and definitive care. In our institution, the decision to initiate haemodialysis is made either by nephrologists or by ICU physicians. In this particular case, the prehospital physician asked for the ICU consultant to be present upon arrival in the ED based on the suspected diagnosis. The patient was therefore quickly assessed by a multidisciplinary team, and therapeutic decisions were rapidly made. All further necessary steps (intubation, ICU admission, dialysis catheter insertion, haemodialysis initiation) were quickly initiated, and the time spent in the ED was therefore limited to a minimum. Metformin-associated metabolic acidosis is a rare but well-known complication of metformin treatment. Its incidence was calculated to be 4.3 cases per 100,000 patient-years [14]. When it develops, mortality rates as high as 48% have been reported [15,16]. Clinically significant metformin intoxication and lactic acid accumulation almost always occur in the presence of comorbid conditions, such as impaired kidney function. In our case, the patient indeed presented a KDIGO stage 3 acute kidney injury due to pre-renal disease consecutive to severe dehydration and sepsis. Prompt identification and treatment are mandatory in such cases, but diagnosis might be particularly difficult because of aspecific presentation. Moreover, acidaemia can be profound even though the patient does not seem to be critically ill. Although the main cause of the lactic acidosis found on ABG analysis was rapidly identified in this case, other potential aetiologies should be always considered. These include severe sepsis, shock, anemia, seizures, hypoxemia, carbon monoxide or cyanide poisoning, ethylene glycol or salicylates intoxication, alcoholism and liver disease, beta-agonist medication or profound thiamine deficit [17]. In our situation, these aetiologies were rapidly excluded by clinical appreciation and laboratory analysis. The role of bicarbonates infusion is still debated in metformin-associated metabolic acidosis as in other causes of anion gap metabolic acidosis [18]. In our case, this treatment was administered given the profound acidaemia and the signs of respiratory exhaustion, with the aim of avoiding endotracheal intubation. As the patient did not improve under supportive care and bicarbonate infusion, the decision to employ extracorporeal removal was swiftly made and, by virtue of a rapid diagnostic path, haemodialysis was started approximately 6 hours after the first medical contact. Haemodialysis has been shown to be effective in cases of metformin-associated metabolic acidoses [19] and is recommended in patients showing signs of severe poisoning. These signs include a lactate concentration higher than 20 mmol/L, a pH ≤ 7.0, or failure to improve with standard treatment within a few hours [4] as in our case. 4. Conclusions This case report shows that prehospital ABG measurement is feasible and can improve diagnostic accuracy in the field. In this particular case, ABG analysis enabled the prehospital physician to make the correct diagnosis in a timely manner, thus allowing the appropriate treatment to be initiated before hospital admission. Unnecessary delay and potential harm to the patient were thereby avoided. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author Contributions Writing—original draft preparation, P.E.F. and L.S.; writing—review and editing, P.E.F. and L.S. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Not applicable. Informed Consent Statement Informed consent was obtained from the patient whose case is reported in this manuscript. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest.	METFORMIN HYDROCHLORIDE	DrugsGivenReaction	CC BY	33466796	18,853,427	2021-01-14
What was the outcome of reaction 'Acute kidney injury'?	Prehospital Diagnosis of Shortness of Breath Caused by Profound Metformin-Associated Metabolic Acidosis. Shortness of breath is a common complaint among patients in emergency medicine. While most common causes are usually promptly identified, less frequent aetiologies might be challenging to diagnose, especially in the pre-hospital setting. We report a case of prehospital dyspnoea initially ascribed to pulmonary oedema which turned out to be the result of profound metformin-associated metabolic acidosis. This diagnosis was already made during the prehospital phase by virtue of arterial blood gas measurement. Pre-hospital measurement of arterial blood gases is therefore feasible and can improve diagnostic accuracy in the field, thus avoiding unnecessary delay and potential harm to the patient before initiating the appropriate therapeutic actions. 1. Introduction Shortness of breath (SOB) is one of the most common complaints among patients in prehospital emergency medicine [1]. Most common causes of shortness of breath (pulmonary oedema, pneumonia, chronic obstructive pulmonary disease exacerbation, pulmonary embolism, pneumothorax, etc.) are usually identified rather easily, although less common aetiologies might be overlooked and therefore misdiagnosed if clinicians fail to consider them. Furthermore, even a high clinical suspicion does not equal a definitive diagnosis, which might require para-clinical confirmation through specific investigations such as arterial blood gas (ABG) analysis. We report the case of a patient complaining of SOB that was initially imputed to pulmonary oedema. However, an ABG analysis performed in the field allowed the prehospital emergency physician to identify a profound metformin-associated metabolic acidosis as the actual cause of the patient’s dyspnoea. 2. Case Description The emergency medical call centre (EMCC) dispatched an advanced life support ambulance to take care of a 71-year-old woman who had called complaining about SOB. Upon arrival, prehospital providers noted shallow breathing with a respiratory rate of over 35 breaths per minute. Lung fields were clear to auscultation and the patient showed no sign of cyanosis. There was no haemodynamic compromise. The Glasgow Coma Scale was 15 with no focal deficit and the patient was normothermic. Given the respiratory distress, paramedics initiated oxygen therapy using a high-flow non-rebreather mask. Suspecting acute heart failure (AHF), they asked the EMCC to dispatch a medical mobile unit called ‘Service Mobile d’Urgence et de Réanimation’ (SMUR). These units are staffed by an advanced paramedic and a physician and can be dispatched to assist an ambulance in case of life-threatening emergencies [2]. These SMURs are the only prehospital units equipped with non-invasive ventilation (NIV) devices and portable blood gas analysers (iStat, Abbott Point of Care Inc., Princeton, NJ, USA) and are therefore often called upon to enhance the management of patients in acute respiratory distress or failure. While awaiting the arrival of the SMUR, the paramedics continued their evaluation and discovered that the patient was under metformin for diabetes and diuretics for hypertension, and that she was on day 3 of antibiotic treatment for urinary tract infection. She did not use cigarettes, alcohol, or illicit drugs. The patient was found to be hypoglycaemic at 2.3 mmol/L and received intravenous glucose accordingly. An ABG analysis performed by the SMUR physician shortly after arriving on site showed severe lactic acidosis (FiO2 65%: pH 6.845, PaCO2 1.95 kPa, PaO2 27.7 kPa, HCO3−—2.5 mmol/L, lactate 17.0 mmol/L). While AHF was suspected at first, the profound metabolic acidosis revealed by the ABG analysis was not compatible with the clinical picture. Indeed, the patient was not in cardiogenic shock, and a diagnosis of lactic acidosis secondary to metformin intoxication was thereafter suspected. Intravenous hydration was started and bicarbonates were administered. Since the patient showed signs of respiratory distress and exhaustion, NIV was initiated. In-hospital investigations confirmed severe lactic acidosis and acute kidney injury KDIGO stage 3 [3], with a creatinine of 788 µmol/L which yielded an estimated glomerular filtration rate of 5 mL/min/m2 according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. This kidney injury was secondary to the pre-renal disease on severe dehydration (diuretics, decreased drinking inputs, etc.) and sepsis, with consequent acute tubular necrosis. The patient showed no sign of hepatic impairment nor hypoxaemia. There was no hint that the patient’s illness could have been secondary to suicidal or accidental biguanide ingestion. The diagnosis of profound metformin-associated metabolic acidosis secondary to renal failure was definitively established at this stage. Despite aggressive hydration and bicarbonates infusion, the patient remained oligo-anuric with severe metabolic acidosis and showed signs of respiratory exhaustion finally requiring endotracheal intubation. She was admitted to the intensive care unit (ICU) for haemodynamic and respiratory management. The decision to start haemodialysis was finally made basing on the severity of acidosis and lactate level, coexisting renal failure and failure to improve with bicarbonate therapy [4]. The patient improved rapidly under this treatment and was extubated on the next day after pH normalisation and lactate clearance. She was transferred to the internal medicine ward after 48 h and finally discharged from the hospital 10 days later, after full renal recovery. 3. Discussion SOB is a major complaint among pre-hospital and ED patients, accounting for over 3.9 million visits in 2017 in the United States [5]. Most of these visits involve patients over 65 years old. Generally, cases of SOB are readily identified and can be ascribed to frequent aetiologies, i.e., heart failure (43%), community-acquired pneumonia (35%), acute exacerbation of chronic respiratory disease (32%), pulmonary embolism (18%), and acute asthma (3%). Two coexisting causes of SOB are found in as many as 47% of patients [6]. Nevertheless, clinicians must be aware that other less frequent aetiologies of SOB should be considered (salicylate intoxication, carbon monoxide poisoning, toxin-related metabolic acidosis, diabetic ketoacidosis, sepsis, anaemia, etc.). They should also acknowledge that diagnosing infrequent causes of SOB might be challenging, especially in the pre-hospital setting, where resources are limited and most investigation tools unavailable. Inappropriate dismissal of infrequent causes of SOB might lead to delayed diagnosis and treatment. Such delays might prove harmful, most particularly in old and frail patients. Our case exemplifies how pre-hospital ABG measurement can easily improve diagnostic accuracy in such cases. In our prehospital system and in many European countries, trained physicians can be dispatched by EMCCs to provide medical care in the field [7], and can therefore draw and analyze ABG. However, even though recent and compact point-of-care testing devices allow for quick and reliable ABG analysis in the field [8], most emergency medical systems still lack such tools in their standard equipment. Barriers to the implementation of ABG analysis devices in the field have scarcely been described, but might include the absence of prehospital emergency physicians to interpret the results and the fear of unnecessarily delaying transport. Prehospital systems unable to dispatch emergency physicians in the field could however develop specific procedures to allow the use of and interpretation of ABG. Paramedics could be trained to draw arterial blood, protocols could be created to help paramedics determine cases in which ABG analysis could be of use, and distance interpretation by medical supervisors could be considered. Similar procedures have been successfully developed regarding electrocardiographic interpretation [9,10]. In the absence of distance medical supervision, regular algorithms or even electronic apps could be developed to facilitate the interpretation of ABG results [11,12]. Unnecessarily delaying transport could be avoided by limiting and clearly identifying the indications to ABG analysis, and by limiting the number of attempts and the time allowed to draw arterial blood. Moreover, other procedures can be performed while blood is drawn and analyzed, thereby further limiting potential delays. In our system, physicians are allowed only one attempt in a maximum of 60 s. Absolute contraindications to ABG sampling can usually be clinically identified easily (local infection, thrombosis or distorted anatomy, severe peripheral vascular disease, abnormal Allen’s test, etc.), and serious complications (infection, arterial occlusion or embolism, nerve injury, pseudo-aneurysm or vessel laceration, etc.) are rare. Minor complications (local pain, minor bleeding or hematoma, vasovagal response, etc.) can be easily managed and anticoagulants or antiplatelet agents do not represent absolute contraindications but often require only increased vascular pressure. Although venous sampling could lead to misdiagnosis, providers can be trained to recognize such samples. In this particular case, onsite ABG analysis allowed the physician to suspect metformin-associated metabolic acidosis and start the appropriate treatment even in the pre-hospital setting. While previous studies have failed to show a general positive effect of prehospital ABG on diagnostic accuracy, it can be of significant benefit in specific situations [13]. Moreover, ABG has been shown to improve treatment quality in critically ill patients [13]. In our system, prehospital physicians notify the Emergency Department (ED) team through the EMCC and can activate specific medical support before arrival. Consultants are thus notified before the patient’s arrival in the ED, thereby accelerating the initiation of appropriate and definitive care. In our institution, the decision to initiate haemodialysis is made either by nephrologists or by ICU physicians. In this particular case, the prehospital physician asked for the ICU consultant to be present upon arrival in the ED based on the suspected diagnosis. The patient was therefore quickly assessed by a multidisciplinary team, and therapeutic decisions were rapidly made. All further necessary steps (intubation, ICU admission, dialysis catheter insertion, haemodialysis initiation) were quickly initiated, and the time spent in the ED was therefore limited to a minimum. Metformin-associated metabolic acidosis is a rare but well-known complication of metformin treatment. Its incidence was calculated to be 4.3 cases per 100,000 patient-years [14]. When it develops, mortality rates as high as 48% have been reported [15,16]. Clinically significant metformin intoxication and lactic acid accumulation almost always occur in the presence of comorbid conditions, such as impaired kidney function. In our case, the patient indeed presented a KDIGO stage 3 acute kidney injury due to pre-renal disease consecutive to severe dehydration and sepsis. Prompt identification and treatment are mandatory in such cases, but diagnosis might be particularly difficult because of aspecific presentation. Moreover, acidaemia can be profound even though the patient does not seem to be critically ill. Although the main cause of the lactic acidosis found on ABG analysis was rapidly identified in this case, other potential aetiologies should be always considered. These include severe sepsis, shock, anemia, seizures, hypoxemia, carbon monoxide or cyanide poisoning, ethylene glycol or salicylates intoxication, alcoholism and liver disease, beta-agonist medication or profound thiamine deficit [17]. In our situation, these aetiologies were rapidly excluded by clinical appreciation and laboratory analysis. The role of bicarbonates infusion is still debated in metformin-associated metabolic acidosis as in other causes of anion gap metabolic acidosis [18]. In our case, this treatment was administered given the profound acidaemia and the signs of respiratory exhaustion, with the aim of avoiding endotracheal intubation. As the patient did not improve under supportive care and bicarbonate infusion, the decision to employ extracorporeal removal was swiftly made and, by virtue of a rapid diagnostic path, haemodialysis was started approximately 6 hours after the first medical contact. Haemodialysis has been shown to be effective in cases of metformin-associated metabolic acidoses [19] and is recommended in patients showing signs of severe poisoning. These signs include a lactate concentration higher than 20 mmol/L, a pH ≤ 7.0, or failure to improve with standard treatment within a few hours [4] as in our case. 4. Conclusions This case report shows that prehospital ABG measurement is feasible and can improve diagnostic accuracy in the field. In this particular case, ABG analysis enabled the prehospital physician to make the correct diagnosis in a timely manner, thus allowing the appropriate treatment to be initiated before hospital admission. Unnecessary delay and potential harm to the patient were thereby avoided. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author Contributions Writing—original draft preparation, P.E.F. and L.S.; writing—review and editing, P.E.F. and L.S. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Not applicable. Informed Consent Statement Informed consent was obtained from the patient whose case is reported in this manuscript. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest.	Recovered	ReactionOutcome	CC BY	33466796	18,853,427	2021-01-14
What was the outcome of reaction 'Dyspnoea'?	Prehospital Diagnosis of Shortness of Breath Caused by Profound Metformin-Associated Metabolic Acidosis. Shortness of breath is a common complaint among patients in emergency medicine. While most common causes are usually promptly identified, less frequent aetiologies might be challenging to diagnose, especially in the pre-hospital setting. We report a case of prehospital dyspnoea initially ascribed to pulmonary oedema which turned out to be the result of profound metformin-associated metabolic acidosis. This diagnosis was already made during the prehospital phase by virtue of arterial blood gas measurement. Pre-hospital measurement of arterial blood gases is therefore feasible and can improve diagnostic accuracy in the field, thus avoiding unnecessary delay and potential harm to the patient before initiating the appropriate therapeutic actions. 1. Introduction Shortness of breath (SOB) is one of the most common complaints among patients in prehospital emergency medicine [1]. Most common causes of shortness of breath (pulmonary oedema, pneumonia, chronic obstructive pulmonary disease exacerbation, pulmonary embolism, pneumothorax, etc.) are usually identified rather easily, although less common aetiologies might be overlooked and therefore misdiagnosed if clinicians fail to consider them. Furthermore, even a high clinical suspicion does not equal a definitive diagnosis, which might require para-clinical confirmation through specific investigations such as arterial blood gas (ABG) analysis. We report the case of a patient complaining of SOB that was initially imputed to pulmonary oedema. However, an ABG analysis performed in the field allowed the prehospital emergency physician to identify a profound metformin-associated metabolic acidosis as the actual cause of the patient’s dyspnoea. 2. Case Description The emergency medical call centre (EMCC) dispatched an advanced life support ambulance to take care of a 71-year-old woman who had called complaining about SOB. Upon arrival, prehospital providers noted shallow breathing with a respiratory rate of over 35 breaths per minute. Lung fields were clear to auscultation and the patient showed no sign of cyanosis. There was no haemodynamic compromise. The Glasgow Coma Scale was 15 with no focal deficit and the patient was normothermic. Given the respiratory distress, paramedics initiated oxygen therapy using a high-flow non-rebreather mask. Suspecting acute heart failure (AHF), they asked the EMCC to dispatch a medical mobile unit called ‘Service Mobile d’Urgence et de Réanimation’ (SMUR). These units are staffed by an advanced paramedic and a physician and can be dispatched to assist an ambulance in case of life-threatening emergencies [2]. These SMURs are the only prehospital units equipped with non-invasive ventilation (NIV) devices and portable blood gas analysers (iStat, Abbott Point of Care Inc., Princeton, NJ, USA) and are therefore often called upon to enhance the management of patients in acute respiratory distress or failure. While awaiting the arrival of the SMUR, the paramedics continued their evaluation and discovered that the patient was under metformin for diabetes and diuretics for hypertension, and that she was on day 3 of antibiotic treatment for urinary tract infection. She did not use cigarettes, alcohol, or illicit drugs. The patient was found to be hypoglycaemic at 2.3 mmol/L and received intravenous glucose accordingly. An ABG analysis performed by the SMUR physician shortly after arriving on site showed severe lactic acidosis (FiO2 65%: pH 6.845, PaCO2 1.95 kPa, PaO2 27.7 kPa, HCO3−—2.5 mmol/L, lactate 17.0 mmol/L). While AHF was suspected at first, the profound metabolic acidosis revealed by the ABG analysis was not compatible with the clinical picture. Indeed, the patient was not in cardiogenic shock, and a diagnosis of lactic acidosis secondary to metformin intoxication was thereafter suspected. Intravenous hydration was started and bicarbonates were administered. Since the patient showed signs of respiratory distress and exhaustion, NIV was initiated. In-hospital investigations confirmed severe lactic acidosis and acute kidney injury KDIGO stage 3 [3], with a creatinine of 788 µmol/L which yielded an estimated glomerular filtration rate of 5 mL/min/m2 according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. This kidney injury was secondary to the pre-renal disease on severe dehydration (diuretics, decreased drinking inputs, etc.) and sepsis, with consequent acute tubular necrosis. The patient showed no sign of hepatic impairment nor hypoxaemia. There was no hint that the patient’s illness could have been secondary to suicidal or accidental biguanide ingestion. The diagnosis of profound metformin-associated metabolic acidosis secondary to renal failure was definitively established at this stage. Despite aggressive hydration and bicarbonates infusion, the patient remained oligo-anuric with severe metabolic acidosis and showed signs of respiratory exhaustion finally requiring endotracheal intubation. She was admitted to the intensive care unit (ICU) for haemodynamic and respiratory management. The decision to start haemodialysis was finally made basing on the severity of acidosis and lactate level, coexisting renal failure and failure to improve with bicarbonate therapy [4]. The patient improved rapidly under this treatment and was extubated on the next day after pH normalisation and lactate clearance. She was transferred to the internal medicine ward after 48 h and finally discharged from the hospital 10 days later, after full renal recovery. 3. Discussion SOB is a major complaint among pre-hospital and ED patients, accounting for over 3.9 million visits in 2017 in the United States [5]. Most of these visits involve patients over 65 years old. Generally, cases of SOB are readily identified and can be ascribed to frequent aetiologies, i.e., heart failure (43%), community-acquired pneumonia (35%), acute exacerbation of chronic respiratory disease (32%), pulmonary embolism (18%), and acute asthma (3%). Two coexisting causes of SOB are found in as many as 47% of patients [6]. Nevertheless, clinicians must be aware that other less frequent aetiologies of SOB should be considered (salicylate intoxication, carbon monoxide poisoning, toxin-related metabolic acidosis, diabetic ketoacidosis, sepsis, anaemia, etc.). They should also acknowledge that diagnosing infrequent causes of SOB might be challenging, especially in the pre-hospital setting, where resources are limited and most investigation tools unavailable. Inappropriate dismissal of infrequent causes of SOB might lead to delayed diagnosis and treatment. Such delays might prove harmful, most particularly in old and frail patients. Our case exemplifies how pre-hospital ABG measurement can easily improve diagnostic accuracy in such cases. In our prehospital system and in many European countries, trained physicians can be dispatched by EMCCs to provide medical care in the field [7], and can therefore draw and analyze ABG. However, even though recent and compact point-of-care testing devices allow for quick and reliable ABG analysis in the field [8], most emergency medical systems still lack such tools in their standard equipment. Barriers to the implementation of ABG analysis devices in the field have scarcely been described, but might include the absence of prehospital emergency physicians to interpret the results and the fear of unnecessarily delaying transport. Prehospital systems unable to dispatch emergency physicians in the field could however develop specific procedures to allow the use of and interpretation of ABG. Paramedics could be trained to draw arterial blood, protocols could be created to help paramedics determine cases in which ABG analysis could be of use, and distance interpretation by medical supervisors could be considered. Similar procedures have been successfully developed regarding electrocardiographic interpretation [9,10]. In the absence of distance medical supervision, regular algorithms or even electronic apps could be developed to facilitate the interpretation of ABG results [11,12]. Unnecessarily delaying transport could be avoided by limiting and clearly identifying the indications to ABG analysis, and by limiting the number of attempts and the time allowed to draw arterial blood. Moreover, other procedures can be performed while blood is drawn and analyzed, thereby further limiting potential delays. In our system, physicians are allowed only one attempt in a maximum of 60 s. Absolute contraindications to ABG sampling can usually be clinically identified easily (local infection, thrombosis or distorted anatomy, severe peripheral vascular disease, abnormal Allen’s test, etc.), and serious complications (infection, arterial occlusion or embolism, nerve injury, pseudo-aneurysm or vessel laceration, etc.) are rare. Minor complications (local pain, minor bleeding or hematoma, vasovagal response, etc.) can be easily managed and anticoagulants or antiplatelet agents do not represent absolute contraindications but often require only increased vascular pressure. Although venous sampling could lead to misdiagnosis, providers can be trained to recognize such samples. In this particular case, onsite ABG analysis allowed the physician to suspect metformin-associated metabolic acidosis and start the appropriate treatment even in the pre-hospital setting. While previous studies have failed to show a general positive effect of prehospital ABG on diagnostic accuracy, it can be of significant benefit in specific situations [13]. Moreover, ABG has been shown to improve treatment quality in critically ill patients [13]. In our system, prehospital physicians notify the Emergency Department (ED) team through the EMCC and can activate specific medical support before arrival. Consultants are thus notified before the patient’s arrival in the ED, thereby accelerating the initiation of appropriate and definitive care. In our institution, the decision to initiate haemodialysis is made either by nephrologists or by ICU physicians. In this particular case, the prehospital physician asked for the ICU consultant to be present upon arrival in the ED based on the suspected diagnosis. The patient was therefore quickly assessed by a multidisciplinary team, and therapeutic decisions were rapidly made. All further necessary steps (intubation, ICU admission, dialysis catheter insertion, haemodialysis initiation) were quickly initiated, and the time spent in the ED was therefore limited to a minimum. Metformin-associated metabolic acidosis is a rare but well-known complication of metformin treatment. Its incidence was calculated to be 4.3 cases per 100,000 patient-years [14]. When it develops, mortality rates as high as 48% have been reported [15,16]. Clinically significant metformin intoxication and lactic acid accumulation almost always occur in the presence of comorbid conditions, such as impaired kidney function. In our case, the patient indeed presented a KDIGO stage 3 acute kidney injury due to pre-renal disease consecutive to severe dehydration and sepsis. Prompt identification and treatment are mandatory in such cases, but diagnosis might be particularly difficult because of aspecific presentation. Moreover, acidaemia can be profound even though the patient does not seem to be critically ill. Although the main cause of the lactic acidosis found on ABG analysis was rapidly identified in this case, other potential aetiologies should be always considered. These include severe sepsis, shock, anemia, seizures, hypoxemia, carbon monoxide or cyanide poisoning, ethylene glycol or salicylates intoxication, alcoholism and liver disease, beta-agonist medication or profound thiamine deficit [17]. In our situation, these aetiologies were rapidly excluded by clinical appreciation and laboratory analysis. The role of bicarbonates infusion is still debated in metformin-associated metabolic acidosis as in other causes of anion gap metabolic acidosis [18]. In our case, this treatment was administered given the profound acidaemia and the signs of respiratory exhaustion, with the aim of avoiding endotracheal intubation. As the patient did not improve under supportive care and bicarbonate infusion, the decision to employ extracorporeal removal was swiftly made and, by virtue of a rapid diagnostic path, haemodialysis was started approximately 6 hours after the first medical contact. Haemodialysis has been shown to be effective in cases of metformin-associated metabolic acidoses [19] and is recommended in patients showing signs of severe poisoning. These signs include a lactate concentration higher than 20 mmol/L, a pH ≤ 7.0, or failure to improve with standard treatment within a few hours [4] as in our case. 4. Conclusions This case report shows that prehospital ABG measurement is feasible and can improve diagnostic accuracy in the field. In this particular case, ABG analysis enabled the prehospital physician to make the correct diagnosis in a timely manner, thus allowing the appropriate treatment to be initiated before hospital admission. Unnecessary delay and potential harm to the patient were thereby avoided. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author Contributions Writing—original draft preparation, P.E.F. and L.S.; writing—review and editing, P.E.F. and L.S. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Not applicable. Informed Consent Statement Informed consent was obtained from the patient whose case is reported in this manuscript. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest.	Recovered	ReactionOutcome	CC BY	33466796	18,855,942	2021-01-14
What was the outcome of reaction 'Metabolic acidosis'?	Prehospital Diagnosis of Shortness of Breath Caused by Profound Metformin-Associated Metabolic Acidosis. Shortness of breath is a common complaint among patients in emergency medicine. While most common causes are usually promptly identified, less frequent aetiologies might be challenging to diagnose, especially in the pre-hospital setting. We report a case of prehospital dyspnoea initially ascribed to pulmonary oedema which turned out to be the result of profound metformin-associated metabolic acidosis. This diagnosis was already made during the prehospital phase by virtue of arterial blood gas measurement. Pre-hospital measurement of arterial blood gases is therefore feasible and can improve diagnostic accuracy in the field, thus avoiding unnecessary delay and potential harm to the patient before initiating the appropriate therapeutic actions. 1. Introduction Shortness of breath (SOB) is one of the most common complaints among patients in prehospital emergency medicine [1]. Most common causes of shortness of breath (pulmonary oedema, pneumonia, chronic obstructive pulmonary disease exacerbation, pulmonary embolism, pneumothorax, etc.) are usually identified rather easily, although less common aetiologies might be overlooked and therefore misdiagnosed if clinicians fail to consider them. Furthermore, even a high clinical suspicion does not equal a definitive diagnosis, which might require para-clinical confirmation through specific investigations such as arterial blood gas (ABG) analysis. We report the case of a patient complaining of SOB that was initially imputed to pulmonary oedema. However, an ABG analysis performed in the field allowed the prehospital emergency physician to identify a profound metformin-associated metabolic acidosis as the actual cause of the patient’s dyspnoea. 2. Case Description The emergency medical call centre (EMCC) dispatched an advanced life support ambulance to take care of a 71-year-old woman who had called complaining about SOB. Upon arrival, prehospital providers noted shallow breathing with a respiratory rate of over 35 breaths per minute. Lung fields were clear to auscultation and the patient showed no sign of cyanosis. There was no haemodynamic compromise. The Glasgow Coma Scale was 15 with no focal deficit and the patient was normothermic. Given the respiratory distress, paramedics initiated oxygen therapy using a high-flow non-rebreather mask. Suspecting acute heart failure (AHF), they asked the EMCC to dispatch a medical mobile unit called ‘Service Mobile d’Urgence et de Réanimation’ (SMUR). These units are staffed by an advanced paramedic and a physician and can be dispatched to assist an ambulance in case of life-threatening emergencies [2]. These SMURs are the only prehospital units equipped with non-invasive ventilation (NIV) devices and portable blood gas analysers (iStat, Abbott Point of Care Inc., Princeton, NJ, USA) and are therefore often called upon to enhance the management of patients in acute respiratory distress or failure. While awaiting the arrival of the SMUR, the paramedics continued their evaluation and discovered that the patient was under metformin for diabetes and diuretics for hypertension, and that she was on day 3 of antibiotic treatment for urinary tract infection. She did not use cigarettes, alcohol, or illicit drugs. The patient was found to be hypoglycaemic at 2.3 mmol/L and received intravenous glucose accordingly. An ABG analysis performed by the SMUR physician shortly after arriving on site showed severe lactic acidosis (FiO2 65%: pH 6.845, PaCO2 1.95 kPa, PaO2 27.7 kPa, HCO3−—2.5 mmol/L, lactate 17.0 mmol/L). While AHF was suspected at first, the profound metabolic acidosis revealed by the ABG analysis was not compatible with the clinical picture. Indeed, the patient was not in cardiogenic shock, and a diagnosis of lactic acidosis secondary to metformin intoxication was thereafter suspected. Intravenous hydration was started and bicarbonates were administered. Since the patient showed signs of respiratory distress and exhaustion, NIV was initiated. In-hospital investigations confirmed severe lactic acidosis and acute kidney injury KDIGO stage 3 [3], with a creatinine of 788 µmol/L which yielded an estimated glomerular filtration rate of 5 mL/min/m2 according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. This kidney injury was secondary to the pre-renal disease on severe dehydration (diuretics, decreased drinking inputs, etc.) and sepsis, with consequent acute tubular necrosis. The patient showed no sign of hepatic impairment nor hypoxaemia. There was no hint that the patient’s illness could have been secondary to suicidal or accidental biguanide ingestion. The diagnosis of profound metformin-associated metabolic acidosis secondary to renal failure was definitively established at this stage. Despite aggressive hydration and bicarbonates infusion, the patient remained oligo-anuric with severe metabolic acidosis and showed signs of respiratory exhaustion finally requiring endotracheal intubation. She was admitted to the intensive care unit (ICU) for haemodynamic and respiratory management. The decision to start haemodialysis was finally made basing on the severity of acidosis and lactate level, coexisting renal failure and failure to improve with bicarbonate therapy [4]. The patient improved rapidly under this treatment and was extubated on the next day after pH normalisation and lactate clearance. She was transferred to the internal medicine ward after 48 h and finally discharged from the hospital 10 days later, after full renal recovery. 3. Discussion SOB is a major complaint among pre-hospital and ED patients, accounting for over 3.9 million visits in 2017 in the United States [5]. Most of these visits involve patients over 65 years old. Generally, cases of SOB are readily identified and can be ascribed to frequent aetiologies, i.e., heart failure (43%), community-acquired pneumonia (35%), acute exacerbation of chronic respiratory disease (32%), pulmonary embolism (18%), and acute asthma (3%). Two coexisting causes of SOB are found in as many as 47% of patients [6]. Nevertheless, clinicians must be aware that other less frequent aetiologies of SOB should be considered (salicylate intoxication, carbon monoxide poisoning, toxin-related metabolic acidosis, diabetic ketoacidosis, sepsis, anaemia, etc.). They should also acknowledge that diagnosing infrequent causes of SOB might be challenging, especially in the pre-hospital setting, where resources are limited and most investigation tools unavailable. Inappropriate dismissal of infrequent causes of SOB might lead to delayed diagnosis and treatment. Such delays might prove harmful, most particularly in old and frail patients. Our case exemplifies how pre-hospital ABG measurement can easily improve diagnostic accuracy in such cases. In our prehospital system and in many European countries, trained physicians can be dispatched by EMCCs to provide medical care in the field [7], and can therefore draw and analyze ABG. However, even though recent and compact point-of-care testing devices allow for quick and reliable ABG analysis in the field [8], most emergency medical systems still lack such tools in their standard equipment. Barriers to the implementation of ABG analysis devices in the field have scarcely been described, but might include the absence of prehospital emergency physicians to interpret the results and the fear of unnecessarily delaying transport. Prehospital systems unable to dispatch emergency physicians in the field could however develop specific procedures to allow the use of and interpretation of ABG. Paramedics could be trained to draw arterial blood, protocols could be created to help paramedics determine cases in which ABG analysis could be of use, and distance interpretation by medical supervisors could be considered. Similar procedures have been successfully developed regarding electrocardiographic interpretation [9,10]. In the absence of distance medical supervision, regular algorithms or even electronic apps could be developed to facilitate the interpretation of ABG results [11,12]. Unnecessarily delaying transport could be avoided by limiting and clearly identifying the indications to ABG analysis, and by limiting the number of attempts and the time allowed to draw arterial blood. Moreover, other procedures can be performed while blood is drawn and analyzed, thereby further limiting potential delays. In our system, physicians are allowed only one attempt in a maximum of 60 s. Absolute contraindications to ABG sampling can usually be clinically identified easily (local infection, thrombosis or distorted anatomy, severe peripheral vascular disease, abnormal Allen’s test, etc.), and serious complications (infection, arterial occlusion or embolism, nerve injury, pseudo-aneurysm or vessel laceration, etc.) are rare. Minor complications (local pain, minor bleeding or hematoma, vasovagal response, etc.) can be easily managed and anticoagulants or antiplatelet agents do not represent absolute contraindications but often require only increased vascular pressure. Although venous sampling could lead to misdiagnosis, providers can be trained to recognize such samples. In this particular case, onsite ABG analysis allowed the physician to suspect metformin-associated metabolic acidosis and start the appropriate treatment even in the pre-hospital setting. While previous studies have failed to show a general positive effect of prehospital ABG on diagnostic accuracy, it can be of significant benefit in specific situations [13]. Moreover, ABG has been shown to improve treatment quality in critically ill patients [13]. In our system, prehospital physicians notify the Emergency Department (ED) team through the EMCC and can activate specific medical support before arrival. Consultants are thus notified before the patient’s arrival in the ED, thereby accelerating the initiation of appropriate and definitive care. In our institution, the decision to initiate haemodialysis is made either by nephrologists or by ICU physicians. In this particular case, the prehospital physician asked for the ICU consultant to be present upon arrival in the ED based on the suspected diagnosis. The patient was therefore quickly assessed by a multidisciplinary team, and therapeutic decisions were rapidly made. All further necessary steps (intubation, ICU admission, dialysis catheter insertion, haemodialysis initiation) were quickly initiated, and the time spent in the ED was therefore limited to a minimum. Metformin-associated metabolic acidosis is a rare but well-known complication of metformin treatment. Its incidence was calculated to be 4.3 cases per 100,000 patient-years [14]. When it develops, mortality rates as high as 48% have been reported [15,16]. Clinically significant metformin intoxication and lactic acid accumulation almost always occur in the presence of comorbid conditions, such as impaired kidney function. In our case, the patient indeed presented a KDIGO stage 3 acute kidney injury due to pre-renal disease consecutive to severe dehydration and sepsis. Prompt identification and treatment are mandatory in such cases, but diagnosis might be particularly difficult because of aspecific presentation. Moreover, acidaemia can be profound even though the patient does not seem to be critically ill. Although the main cause of the lactic acidosis found on ABG analysis was rapidly identified in this case, other potential aetiologies should be always considered. These include severe sepsis, shock, anemia, seizures, hypoxemia, carbon monoxide or cyanide poisoning, ethylene glycol or salicylates intoxication, alcoholism and liver disease, beta-agonist medication or profound thiamine deficit [17]. In our situation, these aetiologies were rapidly excluded by clinical appreciation and laboratory analysis. The role of bicarbonates infusion is still debated in metformin-associated metabolic acidosis as in other causes of anion gap metabolic acidosis [18]. In our case, this treatment was administered given the profound acidaemia and the signs of respiratory exhaustion, with the aim of avoiding endotracheal intubation. As the patient did not improve under supportive care and bicarbonate infusion, the decision to employ extracorporeal removal was swiftly made and, by virtue of a rapid diagnostic path, haemodialysis was started approximately 6 hours after the first medical contact. Haemodialysis has been shown to be effective in cases of metformin-associated metabolic acidoses [19] and is recommended in patients showing signs of severe poisoning. These signs include a lactate concentration higher than 20 mmol/L, a pH ≤ 7.0, or failure to improve with standard treatment within a few hours [4] as in our case. 4. Conclusions This case report shows that prehospital ABG measurement is feasible and can improve diagnostic accuracy in the field. In this particular case, ABG analysis enabled the prehospital physician to make the correct diagnosis in a timely manner, thus allowing the appropriate treatment to be initiated before hospital admission. Unnecessary delay and potential harm to the patient were thereby avoided. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author Contributions Writing—original draft preparation, P.E.F. and L.S.; writing—review and editing, P.E.F. and L.S. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Not applicable. Informed Consent Statement Informed consent was obtained from the patient whose case is reported in this manuscript. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest.	Recovered	ReactionOutcome	CC BY	33466796	18,853,427	2021-01-14
What was the outcome of reaction 'Renal tubular necrosis'?	Prehospital Diagnosis of Shortness of Breath Caused by Profound Metformin-Associated Metabolic Acidosis. Shortness of breath is a common complaint among patients in emergency medicine. While most common causes are usually promptly identified, less frequent aetiologies might be challenging to diagnose, especially in the pre-hospital setting. We report a case of prehospital dyspnoea initially ascribed to pulmonary oedema which turned out to be the result of profound metformin-associated metabolic acidosis. This diagnosis was already made during the prehospital phase by virtue of arterial blood gas measurement. Pre-hospital measurement of arterial blood gases is therefore feasible and can improve diagnostic accuracy in the field, thus avoiding unnecessary delay and potential harm to the patient before initiating the appropriate therapeutic actions. 1. Introduction Shortness of breath (SOB) is one of the most common complaints among patients in prehospital emergency medicine [1]. Most common causes of shortness of breath (pulmonary oedema, pneumonia, chronic obstructive pulmonary disease exacerbation, pulmonary embolism, pneumothorax, etc.) are usually identified rather easily, although less common aetiologies might be overlooked and therefore misdiagnosed if clinicians fail to consider them. Furthermore, even a high clinical suspicion does not equal a definitive diagnosis, which might require para-clinical confirmation through specific investigations such as arterial blood gas (ABG) analysis. We report the case of a patient complaining of SOB that was initially imputed to pulmonary oedema. However, an ABG analysis performed in the field allowed the prehospital emergency physician to identify a profound metformin-associated metabolic acidosis as the actual cause of the patient’s dyspnoea. 2. Case Description The emergency medical call centre (EMCC) dispatched an advanced life support ambulance to take care of a 71-year-old woman who had called complaining about SOB. Upon arrival, prehospital providers noted shallow breathing with a respiratory rate of over 35 breaths per minute. Lung fields were clear to auscultation and the patient showed no sign of cyanosis. There was no haemodynamic compromise. The Glasgow Coma Scale was 15 with no focal deficit and the patient was normothermic. Given the respiratory distress, paramedics initiated oxygen therapy using a high-flow non-rebreather mask. Suspecting acute heart failure (AHF), they asked the EMCC to dispatch a medical mobile unit called ‘Service Mobile d’Urgence et de Réanimation’ (SMUR). These units are staffed by an advanced paramedic and a physician and can be dispatched to assist an ambulance in case of life-threatening emergencies [2]. These SMURs are the only prehospital units equipped with non-invasive ventilation (NIV) devices and portable blood gas analysers (iStat, Abbott Point of Care Inc., Princeton, NJ, USA) and are therefore often called upon to enhance the management of patients in acute respiratory distress or failure. While awaiting the arrival of the SMUR, the paramedics continued their evaluation and discovered that the patient was under metformin for diabetes and diuretics for hypertension, and that she was on day 3 of antibiotic treatment for urinary tract infection. She did not use cigarettes, alcohol, or illicit drugs. The patient was found to be hypoglycaemic at 2.3 mmol/L and received intravenous glucose accordingly. An ABG analysis performed by the SMUR physician shortly after arriving on site showed severe lactic acidosis (FiO2 65%: pH 6.845, PaCO2 1.95 kPa, PaO2 27.7 kPa, HCO3−—2.5 mmol/L, lactate 17.0 mmol/L). While AHF was suspected at first, the profound metabolic acidosis revealed by the ABG analysis was not compatible with the clinical picture. Indeed, the patient was not in cardiogenic shock, and a diagnosis of lactic acidosis secondary to metformin intoxication was thereafter suspected. Intravenous hydration was started and bicarbonates were administered. Since the patient showed signs of respiratory distress and exhaustion, NIV was initiated. In-hospital investigations confirmed severe lactic acidosis and acute kidney injury KDIGO stage 3 [3], with a creatinine of 788 µmol/L which yielded an estimated glomerular filtration rate of 5 mL/min/m2 according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. This kidney injury was secondary to the pre-renal disease on severe dehydration (diuretics, decreased drinking inputs, etc.) and sepsis, with consequent acute tubular necrosis. The patient showed no sign of hepatic impairment nor hypoxaemia. There was no hint that the patient’s illness could have been secondary to suicidal or accidental biguanide ingestion. The diagnosis of profound metformin-associated metabolic acidosis secondary to renal failure was definitively established at this stage. Despite aggressive hydration and bicarbonates infusion, the patient remained oligo-anuric with severe metabolic acidosis and showed signs of respiratory exhaustion finally requiring endotracheal intubation. She was admitted to the intensive care unit (ICU) for haemodynamic and respiratory management. The decision to start haemodialysis was finally made basing on the severity of acidosis and lactate level, coexisting renal failure and failure to improve with bicarbonate therapy [4]. The patient improved rapidly under this treatment and was extubated on the next day after pH normalisation and lactate clearance. She was transferred to the internal medicine ward after 48 h and finally discharged from the hospital 10 days later, after full renal recovery. 3. Discussion SOB is a major complaint among pre-hospital and ED patients, accounting for over 3.9 million visits in 2017 in the United States [5]. Most of these visits involve patients over 65 years old. Generally, cases of SOB are readily identified and can be ascribed to frequent aetiologies, i.e., heart failure (43%), community-acquired pneumonia (35%), acute exacerbation of chronic respiratory disease (32%), pulmonary embolism (18%), and acute asthma (3%). Two coexisting causes of SOB are found in as many as 47% of patients [6]. Nevertheless, clinicians must be aware that other less frequent aetiologies of SOB should be considered (salicylate intoxication, carbon monoxide poisoning, toxin-related metabolic acidosis, diabetic ketoacidosis, sepsis, anaemia, etc.). They should also acknowledge that diagnosing infrequent causes of SOB might be challenging, especially in the pre-hospital setting, where resources are limited and most investigation tools unavailable. Inappropriate dismissal of infrequent causes of SOB might lead to delayed diagnosis and treatment. Such delays might prove harmful, most particularly in old and frail patients. Our case exemplifies how pre-hospital ABG measurement can easily improve diagnostic accuracy in such cases. In our prehospital system and in many European countries, trained physicians can be dispatched by EMCCs to provide medical care in the field [7], and can therefore draw and analyze ABG. However, even though recent and compact point-of-care testing devices allow for quick and reliable ABG analysis in the field [8], most emergency medical systems still lack such tools in their standard equipment. Barriers to the implementation of ABG analysis devices in the field have scarcely been described, but might include the absence of prehospital emergency physicians to interpret the results and the fear of unnecessarily delaying transport. Prehospital systems unable to dispatch emergency physicians in the field could however develop specific procedures to allow the use of and interpretation of ABG. Paramedics could be trained to draw arterial blood, protocols could be created to help paramedics determine cases in which ABG analysis could be of use, and distance interpretation by medical supervisors could be considered. Similar procedures have been successfully developed regarding electrocardiographic interpretation [9,10]. In the absence of distance medical supervision, regular algorithms or even electronic apps could be developed to facilitate the interpretation of ABG results [11,12]. Unnecessarily delaying transport could be avoided by limiting and clearly identifying the indications to ABG analysis, and by limiting the number of attempts and the time allowed to draw arterial blood. Moreover, other procedures can be performed while blood is drawn and analyzed, thereby further limiting potential delays. In our system, physicians are allowed only one attempt in a maximum of 60 s. Absolute contraindications to ABG sampling can usually be clinically identified easily (local infection, thrombosis or distorted anatomy, severe peripheral vascular disease, abnormal Allen’s test, etc.), and serious complications (infection, arterial occlusion or embolism, nerve injury, pseudo-aneurysm or vessel laceration, etc.) are rare. Minor complications (local pain, minor bleeding or hematoma, vasovagal response, etc.) can be easily managed and anticoagulants or antiplatelet agents do not represent absolute contraindications but often require only increased vascular pressure. Although venous sampling could lead to misdiagnosis, providers can be trained to recognize such samples. In this particular case, onsite ABG analysis allowed the physician to suspect metformin-associated metabolic acidosis and start the appropriate treatment even in the pre-hospital setting. While previous studies have failed to show a general positive effect of prehospital ABG on diagnostic accuracy, it can be of significant benefit in specific situations [13]. Moreover, ABG has been shown to improve treatment quality in critically ill patients [13]. In our system, prehospital physicians notify the Emergency Department (ED) team through the EMCC and can activate specific medical support before arrival. Consultants are thus notified before the patient’s arrival in the ED, thereby accelerating the initiation of appropriate and definitive care. In our institution, the decision to initiate haemodialysis is made either by nephrologists or by ICU physicians. In this particular case, the prehospital physician asked for the ICU consultant to be present upon arrival in the ED based on the suspected diagnosis. The patient was therefore quickly assessed by a multidisciplinary team, and therapeutic decisions were rapidly made. All further necessary steps (intubation, ICU admission, dialysis catheter insertion, haemodialysis initiation) were quickly initiated, and the time spent in the ED was therefore limited to a minimum. Metformin-associated metabolic acidosis is a rare but well-known complication of metformin treatment. Its incidence was calculated to be 4.3 cases per 100,000 patient-years [14]. When it develops, mortality rates as high as 48% have been reported [15,16]. Clinically significant metformin intoxication and lactic acid accumulation almost always occur in the presence of comorbid conditions, such as impaired kidney function. In our case, the patient indeed presented a KDIGO stage 3 acute kidney injury due to pre-renal disease consecutive to severe dehydration and sepsis. Prompt identification and treatment are mandatory in such cases, but diagnosis might be particularly difficult because of aspecific presentation. Moreover, acidaemia can be profound even though the patient does not seem to be critically ill. Although the main cause of the lactic acidosis found on ABG analysis was rapidly identified in this case, other potential aetiologies should be always considered. These include severe sepsis, shock, anemia, seizures, hypoxemia, carbon monoxide or cyanide poisoning, ethylene glycol or salicylates intoxication, alcoholism and liver disease, beta-agonist medication or profound thiamine deficit [17]. In our situation, these aetiologies were rapidly excluded by clinical appreciation and laboratory analysis. The role of bicarbonates infusion is still debated in metformin-associated metabolic acidosis as in other causes of anion gap metabolic acidosis [18]. In our case, this treatment was administered given the profound acidaemia and the signs of respiratory exhaustion, with the aim of avoiding endotracheal intubation. As the patient did not improve under supportive care and bicarbonate infusion, the decision to employ extracorporeal removal was swiftly made and, by virtue of a rapid diagnostic path, haemodialysis was started approximately 6 hours after the first medical contact. Haemodialysis has been shown to be effective in cases of metformin-associated metabolic acidoses [19] and is recommended in patients showing signs of severe poisoning. These signs include a lactate concentration higher than 20 mmol/L, a pH ≤ 7.0, or failure to improve with standard treatment within a few hours [4] as in our case. 4. Conclusions This case report shows that prehospital ABG measurement is feasible and can improve diagnostic accuracy in the field. In this particular case, ABG analysis enabled the prehospital physician to make the correct diagnosis in a timely manner, thus allowing the appropriate treatment to be initiated before hospital admission. Unnecessary delay and potential harm to the patient were thereby avoided. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author Contributions Writing—original draft preparation, P.E.F. and L.S.; writing—review and editing, P.E.F. and L.S. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Not applicable. Informed Consent Statement Informed consent was obtained from the patient whose case is reported in this manuscript. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest.	Recovered	ReactionOutcome	CC BY	33466796	18,855,942	2021-01-14
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug-induced liver injury'.	Denosumab-Induced Immune Hepatitis. Denosumab-Prolia®, Xgeva® (Amgen) is a fully human antibody to the receptor activator of the nuclear factor-K ligand (RANKL). Hepatotoxicity is extremely rare, with only one reported case of immune origin. We present a second case of hepatotoxicity resulting from an immune reaction to denosumab. A 43-year-old female was referred to the Endocrinology, Diabetes & Metabolism Department for treatment of low bone mineral density (BMD) following endocrine therapy with letrozole and lucrin because of breast cancer. She developed premature menopause at the age of 36 years when she underwent a left lumpectomy due to an infiltrating duct carcinoma of the breast (T1 NO MO) and was subsequently started on endocrine therapy. Denosumab was started to prevent osteoporosis. On the third year after starting on denosumab and one month after she received the last injection, she became ill. The routine biochemical analysis showed that the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) rose appreciatively to 10 times the upper limit of normal (ULN). The gamma-glutamyl transferase (GGT) level was elevated slightly to 67 U/L (0-38 U/L). The serum gamma-globulin level was elevated to 1.72 g/dL (0.7-1.6 gr/dl), while the total bilirubin (TB) and serum albumin levels were normal. A liver biopsy revealed a moderate to severe chronic inflammatory infiltrate containing MUM-1 positive plasma cells. In addition, numerous CD-3 positive small T lymphocytes and few CD-20 positive B lymphocytes and eosinophils were seen in the portal tracts. Moderate to severe interface hepatitis, bile duct proliferation and mild portal fibrosis were also identified. The results could be consistent with the diagnosis of drug-induced liver injury (DILI). 1. Introduction Denosumab is a human monoclonal antibody targeting the key bone resorption mediator receptor activator of the nuclear factor kappa B (NF-κB) ligand (RANKL). Additionally, denosumab contains odanacatib, a specific inhibitor of the osteoclast protease cathepsin K (OPC-K), and antibodies against the two endogenous inhibitors of bone formation, the proteins sclerostin and dickkopf-1. The drug is administered via subcutaneous injection once every six months and is approved for the treatment of postmenopausal women with osteoporosis at an increased risk of fracture [1]. Denosumab is also one of the first-line treatments recommended for aromatase inhibitor-induced bone loss [2,3]. The onset of menopause is associated with a decrease of the estrogen concentration. The function of the receptor activator of the NF-κB ligand, RANKL, is to support the binding of the receptor activator RANK to the osteoclast [4]. Denosumab binding to RANKL inhibits osteoclast formation, maintenance and survival, and reduces bone resorption and turnover. It has a half-life of 25.4 days. Drug-induced liver injury (DILI) occurs as an unpredicted response to therapeutic doses of a medication (an idiosyncratic reaction–Type B) or as a response to a high dose of the medication (intrinsic toxicity–Type A), which are associated with hepatic injury [5,6]. Type A reactions are a direct result of the drugs’ therapeutic effect. The reaction is dose- and frequency-dependent. Type B reactions are unexpected, unrelated to the dose and frequency of administration, and only occur in a minority of patients who are presumed to have a predisposition [5,6]. In this paper, we present an example of denosumab-induced immune hepatitis. DILI encompasses both acute and/or chronic hepatic lesions. The liver injury may be the only clinical manifestation of the adverse drug effect, or it may be accompanied by injury to other organs or by systemic manifestations. This article supports the safe and effective use of drugs by patients and guides laboratory medicine professionals in determining the possible DILI. We report a second rare case of DILI resulting from exposure to denosumab. 2. Case Report A 43-year-old female was referred to our Endocrinology, Diabetes & Metabolism Department for treatment of low bone mineral density (BMD) following endocrine therapy of breast cancer. She developed premature menopause at the age of 36 years, when she underwent a left lumpectomy due to an infiltrating duct carcinoma of the breast (T1 NO MO). She subsequently underwent adjuvant chemo-radiotherapy. The tumor was estrogen receptor (ER) positive. Due to her history of anticardiolipin syndrome, tamoxifen, a selective ER modulator treatment with an increased risk of thrombosis was contraindicated. She commenced treatment with a combination of an aromatase inhibitor (AI), letrozole, and a gonadotropin-releasing hormone (Gn RH agonist), lucrin, for estrogen suppression. The tablet contains 2.5 mg of letrozole, a nonsteroidal aromatase inhibitor. Femara [4,4′-(1H-1,2,4-Triazol-1-ylmethylene)] inhibits estrogen synthesis. There are no reported drug interactions between lucrin, letrazole or denosumab. The dual-energy X-ray absorptiometry scan T score in the lumbar spine was −2.2, and an oral bisphosphonate was started. In the next year, during bisphosphonate treatment, she developed gastrointestinal symptoms resulting in her treatment being changed to subcutaneous denosumab 60 mg every six months. She also took vitamin D and a calcium replacement. There was no past history of fracture. The patient did not smoke or consume alcohol. The only other therapeutic she took was aspirin. On the third year after starting on denosumab and one month after she had received a subcutaneous administration of the drug, a gradual rise of liver enzymes was noted. The patient became tired and reported a poor appetite. The physical examination was unremarkable. Laboratory investigations revealed a complete blood count (CBC) and international normalized ratio (INR) within normal limits. An elevation of serum transaminases (ALT and AST) to around 10 times ULN was observed. Routine virology tests for the determination of hepatitis virus B (HBV) and virus hepatitis C (HCV), as well as Epstein–Bar virus (EBV) and cytomegalovirus (CMV), were negative. The routine autoimmune serology values for smooth muscle antibody, mitochondrial antibody and anti-nuclear antibody (ANA) were negative. The value for soluble liver antigen-antibody (Ag Ab) was 2.3 (0.0–20.0 units), the value for anti proteinase was 3 < 0.2 (0.00–0.99 index), and the value for anti myeloperoxidase was < 0.2 (0.00–0.99 index). The liver kidney microsome Ab-(LKM, liver kidney microsome andibody) was also negative. There was no evidence of chronic hepatitis. The GGT level was 67 U/L (0–38 U/L). The serum gamma-globulin level was 1.72 g/dL (0.7–1.6 gr/dl). The total bilirubin (TB) and serum albumin levels were normal. An abdominal ultrasound was normal. Scheme 1 shows the longitudinal transaminases data in this patient, from the time she started denosumab, during hospitalization and after stopping the treatment. The levels of the γGT follow the same pattern. As shown in Scheme 2, the enzyme levels spike during the liver toxic episode and decline after the interruption of the therapy. The elevation of this enzyme points to a cholestatic process. 3. Pathology-Methods Samples in formalin were embedded in the Pathology Institute into formalin-fixed paraffin-embedded tissue (FFPE) blocks. The blocks were sectioned by Leica EM2245 microtome into 3–5 µm slices, which were loaded on StarFrost microscope slides for H&E staining or on Leica X-tra Adhesive slides for immunohistochemistry (IHC) staining. Hematoxylin and eosin (H&E) staining slides were loaded onto Sakura’s Tissue-Tek© Prisma & Film for the automatic staining and covering of the slide. The IHC staining protocol includes: slides’ incubation at 60 °C for 45 min, xylen incubation for removal of paraffin, hematoxilyn (PRC LTd., 17610, Harris, Jefferson, OR, USA) incubation for 10 min (two washes for 5 min each) incubation in 70% ethanol for washing, 10 min of incubation in Eosin (Pioneer Research Chemical Ltd., 18553, Colchester, UK) staining, two washings × 5 min incubation with 70% ethanol, 2 washings × 5 min incubation in 100% ETOH, three times × 5 min in Xylen and cover slipping with Xylen film. All procedures were done according to the company’s protocol & recommendations. To read the fibrin deposits, Masson trychrome staining was employed. The slides were loaded on Ultra Bench Ventana and stained according to the validated protocol with cytokeratin (CK)7 Antibody (DAKO, Clone: OVTL12/30, Dil: 1:100, UltraView, standard, 32 min Ab incubation). An additional slide was stained with MUM1 Ab (clone: MUM1p; Dil: 1:50; DBS; Ultra View standard, 37 °C, 42′ + Amplifier). Stains were performed with a Ventana Ultra Bench stain apparatus using an ultraView Universal DAB Detection Kit (Catalog Number: 760). 4. Histology A liver biopsy was performed, which revealed a moderate to severe chronic inflammatory infiltrate. Specifically, the liver core biopsy revealed a well-preserved liver architecture. The parenchyma had a few foci of inflammation. In the portal tracts, there was a moderate to severe infiltrate containing plasma cells, numerous positive CD3+ small T lymphocytes, and few C 20 positive B lymphocytes and eosinophils (Figure 1, Figure 2 and Figure 3). Hyperplasia and mild portal fibrosis were identified using Masson staining (Figure 4). Immunostaining revealed a severe interface hepatitis with bile ductular proliferation on CK-7 staining (Figure 5). Immunohistochemistry (Figure 6) presents an infiltrate containing MUM-1 positive plasma cells in the portal tracts. The likely diagnosis was considered to be consistent with immune hepatitis due to DILI. 5. Discussion Osteoporosis. is characterized by low bone mass, microarchitectural disruption and skeletal fragility, resulting in decreased bone strength and increased risk of fracture. Endocrine therapy, an adjuvant treatment in the management of patients with hormone-sensitive breast cancer is associated with adverse effects on the musculoskeletal system and commonly increases the risk of osteoporosis and fractures. Angiogenesis and osteogenesis connect for the bone function. Blood vessels supply oxygen, nutrients and progenitor cells to the osteogenic environment. The functional vasculature system cooperates for physiological bone healing [7]. The identification of novel therapeutic targets to prevent the disease aimed at inhibiting bone resorption and increasing bone formation. In our patient Denosumab was prescribed to treat aromatase induced bone loss. The combination of the clinical illness together with biochemical abnormalities, the temporal association and the lack of any other suspects remains highly suspicious for denosumab-induced toxic events. The calculated score of 6 (a probable drug-induced adverse reaction) was obtained using the Naranjo score [8]. A causality assessment via the updated RUCAM was done [9]. The updated RUCAM takes into account divergent laboratory analyses of liver injury and classifies it into two different subscales: the hepatocellular type of injury and the cholestatic or mixed type of injury. These types can be differentiated using the ratio R, calculated as the ALT/ALP activity measured at the time of liver injury and expressed as a multiple of ULN. In our case, the liver injury was consistent with hepatocellular and cholestatic injury, and the RUCAM score was 4, leading to a possible causality level of DILI from denosumab. It appears unlikely that a direct hepatotoxic effect would appear after three years of use, and thus the possibility of an immune-mediated drug-induced liver injury was considered more likely. Oral steroid treatment with budesonide at 9 mg/day was commenced. Over the course of the following months, there was a slow decline in the levels of the transaminases and the GGT. Currently the liver tests are normalized, and the patient remains in good health on maintenance therapy with steroids. Denosumab is an effective antiresorptive drug; however, discontinuation of the drug can result in an accelerated bone turnover, rapid loss of bone mineral density and an increased rate of multiple vertebral fractures. However, since the hepatic illness may recur upon reintroduction of the offending drug, the rechallenge of denosumab was not considered ethically possible, especially in view of its prolonged half-life. After denosumab was stopped, treatment was switched to intravenous zolendronic acid at 5 mg/year. The last dual-energy X-ray absorptiometry scan T score was −2.1 at the lumbar spine (BMD 0.925g/cm2), −1.6 at the neck of the femur (BMD 0.817 g/cm2) and −0.6 at the forearm (BMD 0.638 g/cm2). We describe a case of a patient following a lumpectomy because of a duct carcinoma of the breast. Since the tumor expressed a hormone receptor, she was started on endocrine therapy. Consistent with recommendations from several international expert groups [2,3,4,5,6,7,8,10] for the assessment of aromatase inhibitor-induced bone loss, an antiresorptive treatment with oral biphosphonate was started. During the follow-up visits, the bisphosphonate therapy was switched to denosumab. On the third year after starting on denosumab, a gradual rise of liver enzymes was noted. The abnormal liver testing and the result of the liver biopsy suggested a diagnosis of DILI. The temporal relation, the prolonged half-life of the drug and the absence of other causes implicate denosumab as the possible cause of hepatotoxicity, so the drug was stopped. We believe that the liver damage was immune-related due to denosumab. The prolonged steroid treatment needed for the liver disease adds another factor with deleterious effects on the musculoskeletal system and increases the risk of osteoporosis and fractures in our patient. For the preservation of bone health, multiple risk factors are taken into consideration. The treatment varies from basic bone protective measures to specific antiresorptive therapy, and the treatment duration should last for as long as the course of the endocrine therapy is given [11]. For this reason, and because the rechallenge of denosumab was not considered ethically possible, it was extremely important to demonstrate the causality of the drug. Since the liver has a dual blood supply, both systemic and portal, many drugs can cause hepatotoxicity, including those affecting the musculo-skeletal system [12]. Menopause results in a decrease in estrogen concentrations and consequently an enhanced osteoclast maturation and activity. In addition, there is a decrease in the osteoblast function. RANKL and the osteoprotegerin (OPG) system have central roles in the the osteoclast activity, RANKL is expressed on both preosteoblast and stromatolite cell surfaces and plays a central role in osteoclastogenesis by binding to the RANK receptor. RANKL plays an important role in osteoclast differentiation and activation and also in the inhibition of osteoclast apoptosis. OPG, secreted by both osteoblasts and stromatolite cells in the bone marrow, is a soluble member of the tumor necrosis factor superfamily that decreases the RANKL activity and thus inhibits osteoclasts’ differentiation, resulting in a decrease in the osteoclast activity [11]. Since Prolia was approved by the FDA in 2010 for the treatment of osteoporosis, it has been implicated in a large number of clinical trials. Denosumab was not associated with changes in serum aminotransferase levels, and the rates of adverse reactions were similar in patients who received denosumab or placebo. A study population has been published in 2006. The authors described 412 postmenopausal women with a low bone density treated with denosumab [three doses every three or six months], versus alendronate or placebo for 12 months. The bone density increased with denosumab and alendronate but not with placebo. The authors did not find changes in the blood chemistry results [4]. Lewieck et al. [13] followed up the 412 women treated with denosumab, alendronate or a placebo. The incidence of side-effects was similar in all three groups and did not change during the second year of the study. There were “no clinically relevant changes in either serum chemistry or hematology values” [13]. Another study of 255 women with breast cancer and bone metastases treated with denosumab for at least 13 weeks found no “unexpected” changes in liver enzymes [14]. Cummings et al. (2009) reported a randomized placebo-controlled trial of 7868 postmenopausal women with osteoporosis treated with denosumab or a placebo [every six months] for three years. Bone minerals increased and there were fewer fractures in the denosumab-treated patients. The adverse events were similar except for eczema [3% vs. 1.7%], cellulitis [0.3% vs. <0.1%] and flatulence [2.2% vs. 1.4%]. The authors did not describe ALT elevations or hepatotoxicity [15]. Bone et al. (2013) followed 3547 postmenopausal women with osteoporosis treated with denosumab for three or six years. The authors declared that the side-effects did not increase with time; they did not note transaminase elevations or DILI [16]. An additional study described no hepatotoxicity in 4550 postmenopausal women with osteoporosis that were treated for another seven years with denosumab. Serious adverse events included osteonecrosis of the jaw (n = 13) and atypical fractures, but there was no mention of ALT elevations. In their systematic review and meta-analysis where the authors described the efficacy and safety of denosumab in the therapy of bone metastases, they did not mention ALT elevations or hepatotoxicity [17]. Moreover, the Drug Induced Liver Injury Network (DILIN) group reported that among 899 cases of drug-induced liver injury enrolled in a US prospective study between 2004 and 2013, no cases were attributed to denosumab or other agents used to treat osteoporosis [18]. The only reported case of DILI related to Prolia has been reported by our group at Kaplan Medical Center [7]. In this case, to identify the correlation between denosumab and the patient’s liver toxicity, the authors examined the temporal relationship, the amount of drug administered and the duration of the treatment with denosumab. Exposure to over-the-counter medications and both herbal and dietary supplements was ruled out. A lymphocyte toxicity assay (LTA) was performed to determine if denosumab was involved in the liver injury. LTA is based upon incubating the lymphocytes of the specific individual with the drug that produced the reaction (Denosumab) in the presence or absence of a cytochrome P450 system. After 24 h, the lymphocytes are exposed to the yellow tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The LTA of the patient indicated 31% toxicity upon incubation with the monoclonal antibody vs. control. This result implicated denosumab possibly being responsible for the liver reaction. Primary biliary cholangitis and other chronic liver diseases are associated with osteoporosis. There are reports showing lower levels of RANKL in these diseases than in controls [19]. More than this, circulating mononuclear cells could have a higher capacity to differentiate into osteoclasts in patients with chronic liver disease and osteopenia [20]. Presently, Osteoporosis Canada Clinical Practice Guidelines recommend this drug as a first-line therapy for postmenopausal osteoporosis [21]. Wensel’s group summarized serious denosumab-induced infections, dermatologic adverse reactions, as well as possible hypocalcemia [22]. Adverse effects to denosumab are reported in dentistry. A 74-year-old woman treated with denosumab developed osteonecrosis of the mandible [23]. In a recent review article [24], the authors studied bone biopsies collected from patients exposed to denosumab that presented infectious, inflammatory and necrotic jaw diseases. They found different degrees of bone histological changes. However, none of the individuals presented hepatic disturbances [24]. In our first description of denosumab-induced DILI [7], we demonstrated a possible mechanism by which denosumab could cause severe liver toxicity: by blocking and actively reducing RANKL under normal blood levels. In the first report, we described a 72-year-old woman. The liver biopsy revealed a sub-massive hepatic necrosis consistent with DILI. It was suggested that denosumab might produce a cholestatic reaction. The drug also induced the elevation of members of the TNF family and inflammatory chemokines. Because of the prolonged half-life of denosumab (25.4 days), this immune activation can be a long-lasting event. In the present case, the person is also a woman, but she is younger. It is not clear why the onset of the DILI occurred so long after the initiation of Prolia therapy. This suggests immunological priming and, together with the response to steroids, is consistent with an immune mechanism and not autoimmune hepatitis. There is a single report of the production of denosumab-binding antibodies. Two patients who were treated with 100 mg of denosumab administered every six months developed such antibodies, after a month of treatment in one patient and after 12 months in the other. Upon a bioassay evaluation, these were not found to be neutralizing antibodies. They were not detected in subsequent samples [4]. The long half-life of denosumab may play a role in the development of DILI, with a prolonged exposure at low doses. Denosumab reported worldwide sales of $701 million in the third quarter of 2020. It is imperative to follow up on patients for a longer period of time in order to ensure the safety of this drug. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author Contributions V.O. and S.M. took care of the patient during her hospitalization, S.I.-S., A.Y. and N.Z.S. performed the pathological determinations at Kaplan Medical Center in Rehovot, Israel. M.N. performed all the biochemical and immuno-genetic analyses. She also uses her expertise to provide a pharma-toxicological assessment of the case. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. The laboratory analysis and the medical support was part of the standard of care offered by the hospital. Neuman’s work was funded by In Vitro Drug Safety and Biotechnology. Institutional Review Board Statement The patient was treated during her hospitalization at Kaplan Medical Center in Rehovot, Israel. The biopsy and the blood of the patient was taken during her hospitalization and analyzed in the laboratory. All the procedures were part of her standard of care. As a result ethical review and approval were waived for this study. Informed Consent Statement The patient did not need to sign an informed consent since she received the standard of care in the Kaplan Hospital. She agreed in to the presentation of the case report as a scientific publication. Data Availability Statement Data supporting reported results can be found at the hospital recording database. Conflicts of Interest The authors declare no conflict of interest. All the authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All the authors contributed to the study concept and design: acquisition, analysis, and interpretation of data and drafting of the manuscript and revision of the manuscript for intellectual content. Technical support was provided by In Vitro Drug Safety and Biotechnology. Abbreviations AI aromatase inhibitor ALT alanine aminotransferase (glutamic-pyruvic transaminase, SGPT) ALP alkaline phosphatase ANA antinuclear antibody AST aspartate aminotransferase (glutamic-oxalo-acetic transaminase, SGOT) BMD bone mineral density CK cytokeratin CBC compleate blood count CMV cytomegalo-virus DILI drug-induced liver injury EBV Epstein–Bar virus ER estrogen receptor FASLG FAS Ligand–tumor necrosis factor SF6 (TNF-SF6), CD95L/APO1L FFPE Formalin-fixed paraffin-embedded tissue GGT-γ glutamyl-transferase GnRh gonadotropin releasing hormone HBV hepatitis virus B HCV hepatitis virus C H & E Haematoxylen-Eosin IL Interleukin: IL1-α IL1β, IL2, IL4, IL6, IL8, IL 15 INR International Normalized Ratio LTA lymphocyte toxicity assay MTT 3-(4,5-dimethyl-thiazol-2-yl)-2,5-dio-phenyl tetrazolium bromide NFκB nuclear factor-κB OPC-K osteoclast protease cathepsin K OPG osteo-protegerin RANKL receptor activator of nuclear factor-κB ligand TB Total Bilirubin (summation of conjugated and nonconjugated serum bilirubin) ULN Upper Limit of the Normal reference range Figures and Schemes biomedicines-09-00076-sch001_Scheme 1Scheme 1 ALT and AST kinetics. biomedicines-09-00076-sch002_Scheme 2Scheme 2 Kinetics of γGT. Figure 1 H&E staining, plasma cells infiltrate (see in the circle). Figure 2 H&E staining showing lobular inflammation in the portal area. Figure 3 H&E staining showing portal inflammation & inter-phase hepatitis. Figure 4 Masson Trichome staining showing fibrosis of the portal tracts (right top corner). Figure 5 CytoKeratin 7 (CK7) staining showing bile ductular proliferation in the portal tract (marked by the blue square). Figure 6 Positive MUM-1 of plasma cells in the portal tract.	ASPIRIN, CALCIUM, DENOSUMAB, LETROZOLE, LEUPROLIDE ACETATE	DrugsGivenReaction	CC BY	33466886	18,819,705	2021-01-14
What was the administration route of drug 'DENOSUMAB'?	Denosumab-Induced Immune Hepatitis. Denosumab-Prolia®, Xgeva® (Amgen) is a fully human antibody to the receptor activator of the nuclear factor-K ligand (RANKL). Hepatotoxicity is extremely rare, with only one reported case of immune origin. We present a second case of hepatotoxicity resulting from an immune reaction to denosumab. A 43-year-old female was referred to the Endocrinology, Diabetes & Metabolism Department for treatment of low bone mineral density (BMD) following endocrine therapy with letrozole and lucrin because of breast cancer. She developed premature menopause at the age of 36 years when she underwent a left lumpectomy due to an infiltrating duct carcinoma of the breast (T1 NO MO) and was subsequently started on endocrine therapy. Denosumab was started to prevent osteoporosis. On the third year after starting on denosumab and one month after she received the last injection, she became ill. The routine biochemical analysis showed that the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) rose appreciatively to 10 times the upper limit of normal (ULN). The gamma-glutamyl transferase (GGT) level was elevated slightly to 67 U/L (0-38 U/L). The serum gamma-globulin level was elevated to 1.72 g/dL (0.7-1.6 gr/dl), while the total bilirubin (TB) and serum albumin levels were normal. A liver biopsy revealed a moderate to severe chronic inflammatory infiltrate containing MUM-1 positive plasma cells. In addition, numerous CD-3 positive small T lymphocytes and few CD-20 positive B lymphocytes and eosinophils were seen in the portal tracts. Moderate to severe interface hepatitis, bile duct proliferation and mild portal fibrosis were also identified. The results could be consistent with the diagnosis of drug-induced liver injury (DILI). 1. Introduction Denosumab is a human monoclonal antibody targeting the key bone resorption mediator receptor activator of the nuclear factor kappa B (NF-κB) ligand (RANKL). Additionally, denosumab contains odanacatib, a specific inhibitor of the osteoclast protease cathepsin K (OPC-K), and antibodies against the two endogenous inhibitors of bone formation, the proteins sclerostin and dickkopf-1. The drug is administered via subcutaneous injection once every six months and is approved for the treatment of postmenopausal women with osteoporosis at an increased risk of fracture [1]. Denosumab is also one of the first-line treatments recommended for aromatase inhibitor-induced bone loss [2,3]. The onset of menopause is associated with a decrease of the estrogen concentration. The function of the receptor activator of the NF-κB ligand, RANKL, is to support the binding of the receptor activator RANK to the osteoclast [4]. Denosumab binding to RANKL inhibits osteoclast formation, maintenance and survival, and reduces bone resorption and turnover. It has a half-life of 25.4 days. Drug-induced liver injury (DILI) occurs as an unpredicted response to therapeutic doses of a medication (an idiosyncratic reaction–Type B) or as a response to a high dose of the medication (intrinsic toxicity–Type A), which are associated with hepatic injury [5,6]. Type A reactions are a direct result of the drugs’ therapeutic effect. The reaction is dose- and frequency-dependent. Type B reactions are unexpected, unrelated to the dose and frequency of administration, and only occur in a minority of patients who are presumed to have a predisposition [5,6]. In this paper, we present an example of denosumab-induced immune hepatitis. DILI encompasses both acute and/or chronic hepatic lesions. The liver injury may be the only clinical manifestation of the adverse drug effect, or it may be accompanied by injury to other organs or by systemic manifestations. This article supports the safe and effective use of drugs by patients and guides laboratory medicine professionals in determining the possible DILI. We report a second rare case of DILI resulting from exposure to denosumab. 2. Case Report A 43-year-old female was referred to our Endocrinology, Diabetes & Metabolism Department for treatment of low bone mineral density (BMD) following endocrine therapy of breast cancer. She developed premature menopause at the age of 36 years, when she underwent a left lumpectomy due to an infiltrating duct carcinoma of the breast (T1 NO MO). She subsequently underwent adjuvant chemo-radiotherapy. The tumor was estrogen receptor (ER) positive. Due to her history of anticardiolipin syndrome, tamoxifen, a selective ER modulator treatment with an increased risk of thrombosis was contraindicated. She commenced treatment with a combination of an aromatase inhibitor (AI), letrozole, and a gonadotropin-releasing hormone (Gn RH agonist), lucrin, for estrogen suppression. The tablet contains 2.5 mg of letrozole, a nonsteroidal aromatase inhibitor. Femara [4,4′-(1H-1,2,4-Triazol-1-ylmethylene)] inhibits estrogen synthesis. There are no reported drug interactions between lucrin, letrazole or denosumab. The dual-energy X-ray absorptiometry scan T score in the lumbar spine was −2.2, and an oral bisphosphonate was started. In the next year, during bisphosphonate treatment, she developed gastrointestinal symptoms resulting in her treatment being changed to subcutaneous denosumab 60 mg every six months. She also took vitamin D and a calcium replacement. There was no past history of fracture. The patient did not smoke or consume alcohol. The only other therapeutic she took was aspirin. On the third year after starting on denosumab and one month after she had received a subcutaneous administration of the drug, a gradual rise of liver enzymes was noted. The patient became tired and reported a poor appetite. The physical examination was unremarkable. Laboratory investigations revealed a complete blood count (CBC) and international normalized ratio (INR) within normal limits. An elevation of serum transaminases (ALT and AST) to around 10 times ULN was observed. Routine virology tests for the determination of hepatitis virus B (HBV) and virus hepatitis C (HCV), as well as Epstein–Bar virus (EBV) and cytomegalovirus (CMV), were negative. The routine autoimmune serology values for smooth muscle antibody, mitochondrial antibody and anti-nuclear antibody (ANA) were negative. The value for soluble liver antigen-antibody (Ag Ab) was 2.3 (0.0–20.0 units), the value for anti proteinase was 3 < 0.2 (0.00–0.99 index), and the value for anti myeloperoxidase was < 0.2 (0.00–0.99 index). The liver kidney microsome Ab-(LKM, liver kidney microsome andibody) was also negative. There was no evidence of chronic hepatitis. The GGT level was 67 U/L (0–38 U/L). The serum gamma-globulin level was 1.72 g/dL (0.7–1.6 gr/dl). The total bilirubin (TB) and serum albumin levels were normal. An abdominal ultrasound was normal. Scheme 1 shows the longitudinal transaminases data in this patient, from the time she started denosumab, during hospitalization and after stopping the treatment. The levels of the γGT follow the same pattern. As shown in Scheme 2, the enzyme levels spike during the liver toxic episode and decline after the interruption of the therapy. The elevation of this enzyme points to a cholestatic process. 3. Pathology-Methods Samples in formalin were embedded in the Pathology Institute into formalin-fixed paraffin-embedded tissue (FFPE) blocks. The blocks were sectioned by Leica EM2245 microtome into 3–5 µm slices, which were loaded on StarFrost microscope slides for H&E staining or on Leica X-tra Adhesive slides for immunohistochemistry (IHC) staining. Hematoxylin and eosin (H&E) staining slides were loaded onto Sakura’s Tissue-Tek© Prisma & Film for the automatic staining and covering of the slide. The IHC staining protocol includes: slides’ incubation at 60 °C for 45 min, xylen incubation for removal of paraffin, hematoxilyn (PRC LTd., 17610, Harris, Jefferson, OR, USA) incubation for 10 min (two washes for 5 min each) incubation in 70% ethanol for washing, 10 min of incubation in Eosin (Pioneer Research Chemical Ltd., 18553, Colchester, UK) staining, two washings × 5 min incubation with 70% ethanol, 2 washings × 5 min incubation in 100% ETOH, three times × 5 min in Xylen and cover slipping with Xylen film. All procedures were done according to the company’s protocol & recommendations. To read the fibrin deposits, Masson trychrome staining was employed. The slides were loaded on Ultra Bench Ventana and stained according to the validated protocol with cytokeratin (CK)7 Antibody (DAKO, Clone: OVTL12/30, Dil: 1:100, UltraView, standard, 32 min Ab incubation). An additional slide was stained with MUM1 Ab (clone: MUM1p; Dil: 1:50; DBS; Ultra View standard, 37 °C, 42′ + Amplifier). Stains were performed with a Ventana Ultra Bench stain apparatus using an ultraView Universal DAB Detection Kit (Catalog Number: 760). 4. Histology A liver biopsy was performed, which revealed a moderate to severe chronic inflammatory infiltrate. Specifically, the liver core biopsy revealed a well-preserved liver architecture. The parenchyma had a few foci of inflammation. In the portal tracts, there was a moderate to severe infiltrate containing plasma cells, numerous positive CD3+ small T lymphocytes, and few C 20 positive B lymphocytes and eosinophils (Figure 1, Figure 2 and Figure 3). Hyperplasia and mild portal fibrosis were identified using Masson staining (Figure 4). Immunostaining revealed a severe interface hepatitis with bile ductular proliferation on CK-7 staining (Figure 5). Immunohistochemistry (Figure 6) presents an infiltrate containing MUM-1 positive plasma cells in the portal tracts. The likely diagnosis was considered to be consistent with immune hepatitis due to DILI. 5. Discussion Osteoporosis. is characterized by low bone mass, microarchitectural disruption and skeletal fragility, resulting in decreased bone strength and increased risk of fracture. Endocrine therapy, an adjuvant treatment in the management of patients with hormone-sensitive breast cancer is associated with adverse effects on the musculoskeletal system and commonly increases the risk of osteoporosis and fractures. Angiogenesis and osteogenesis connect for the bone function. Blood vessels supply oxygen, nutrients and progenitor cells to the osteogenic environment. The functional vasculature system cooperates for physiological bone healing [7]. The identification of novel therapeutic targets to prevent the disease aimed at inhibiting bone resorption and increasing bone formation. In our patient Denosumab was prescribed to treat aromatase induced bone loss. The combination of the clinical illness together with biochemical abnormalities, the temporal association and the lack of any other suspects remains highly suspicious for denosumab-induced toxic events. The calculated score of 6 (a probable drug-induced adverse reaction) was obtained using the Naranjo score [8]. A causality assessment via the updated RUCAM was done [9]. The updated RUCAM takes into account divergent laboratory analyses of liver injury and classifies it into two different subscales: the hepatocellular type of injury and the cholestatic or mixed type of injury. These types can be differentiated using the ratio R, calculated as the ALT/ALP activity measured at the time of liver injury and expressed as a multiple of ULN. In our case, the liver injury was consistent with hepatocellular and cholestatic injury, and the RUCAM score was 4, leading to a possible causality level of DILI from denosumab. It appears unlikely that a direct hepatotoxic effect would appear after three years of use, and thus the possibility of an immune-mediated drug-induced liver injury was considered more likely. Oral steroid treatment with budesonide at 9 mg/day was commenced. Over the course of the following months, there was a slow decline in the levels of the transaminases and the GGT. Currently the liver tests are normalized, and the patient remains in good health on maintenance therapy with steroids. Denosumab is an effective antiresorptive drug; however, discontinuation of the drug can result in an accelerated bone turnover, rapid loss of bone mineral density and an increased rate of multiple vertebral fractures. However, since the hepatic illness may recur upon reintroduction of the offending drug, the rechallenge of denosumab was not considered ethically possible, especially in view of its prolonged half-life. After denosumab was stopped, treatment was switched to intravenous zolendronic acid at 5 mg/year. The last dual-energy X-ray absorptiometry scan T score was −2.1 at the lumbar spine (BMD 0.925g/cm2), −1.6 at the neck of the femur (BMD 0.817 g/cm2) and −0.6 at the forearm (BMD 0.638 g/cm2). We describe a case of a patient following a lumpectomy because of a duct carcinoma of the breast. Since the tumor expressed a hormone receptor, she was started on endocrine therapy. Consistent with recommendations from several international expert groups [2,3,4,5,6,7,8,10] for the assessment of aromatase inhibitor-induced bone loss, an antiresorptive treatment with oral biphosphonate was started. During the follow-up visits, the bisphosphonate therapy was switched to denosumab. On the third year after starting on denosumab, a gradual rise of liver enzymes was noted. The abnormal liver testing and the result of the liver biopsy suggested a diagnosis of DILI. The temporal relation, the prolonged half-life of the drug and the absence of other causes implicate denosumab as the possible cause of hepatotoxicity, so the drug was stopped. We believe that the liver damage was immune-related due to denosumab. The prolonged steroid treatment needed for the liver disease adds another factor with deleterious effects on the musculoskeletal system and increases the risk of osteoporosis and fractures in our patient. For the preservation of bone health, multiple risk factors are taken into consideration. The treatment varies from basic bone protective measures to specific antiresorptive therapy, and the treatment duration should last for as long as the course of the endocrine therapy is given [11]. For this reason, and because the rechallenge of denosumab was not considered ethically possible, it was extremely important to demonstrate the causality of the drug. Since the liver has a dual blood supply, both systemic and portal, many drugs can cause hepatotoxicity, including those affecting the musculo-skeletal system [12]. Menopause results in a decrease in estrogen concentrations and consequently an enhanced osteoclast maturation and activity. In addition, there is a decrease in the osteoblast function. RANKL and the osteoprotegerin (OPG) system have central roles in the the osteoclast activity, RANKL is expressed on both preosteoblast and stromatolite cell surfaces and plays a central role in osteoclastogenesis by binding to the RANK receptor. RANKL plays an important role in osteoclast differentiation and activation and also in the inhibition of osteoclast apoptosis. OPG, secreted by both osteoblasts and stromatolite cells in the bone marrow, is a soluble member of the tumor necrosis factor superfamily that decreases the RANKL activity and thus inhibits osteoclasts’ differentiation, resulting in a decrease in the osteoclast activity [11]. Since Prolia was approved by the FDA in 2010 for the treatment of osteoporosis, it has been implicated in a large number of clinical trials. Denosumab was not associated with changes in serum aminotransferase levels, and the rates of adverse reactions were similar in patients who received denosumab or placebo. A study population has been published in 2006. The authors described 412 postmenopausal women with a low bone density treated with denosumab [three doses every three or six months], versus alendronate or placebo for 12 months. The bone density increased with denosumab and alendronate but not with placebo. The authors did not find changes in the blood chemistry results [4]. Lewieck et al. [13] followed up the 412 women treated with denosumab, alendronate or a placebo. The incidence of side-effects was similar in all three groups and did not change during the second year of the study. There were “no clinically relevant changes in either serum chemistry or hematology values” [13]. Another study of 255 women with breast cancer and bone metastases treated with denosumab for at least 13 weeks found no “unexpected” changes in liver enzymes [14]. Cummings et al. (2009) reported a randomized placebo-controlled trial of 7868 postmenopausal women with osteoporosis treated with denosumab or a placebo [every six months] for three years. Bone minerals increased and there were fewer fractures in the denosumab-treated patients. The adverse events were similar except for eczema [3% vs. 1.7%], cellulitis [0.3% vs. <0.1%] and flatulence [2.2% vs. 1.4%]. The authors did not describe ALT elevations or hepatotoxicity [15]. Bone et al. (2013) followed 3547 postmenopausal women with osteoporosis treated with denosumab for three or six years. The authors declared that the side-effects did not increase with time; they did not note transaminase elevations or DILI [16]. An additional study described no hepatotoxicity in 4550 postmenopausal women with osteoporosis that were treated for another seven years with denosumab. Serious adverse events included osteonecrosis of the jaw (n = 13) and atypical fractures, but there was no mention of ALT elevations. In their systematic review and meta-analysis where the authors described the efficacy and safety of denosumab in the therapy of bone metastases, they did not mention ALT elevations or hepatotoxicity [17]. Moreover, the Drug Induced Liver Injury Network (DILIN) group reported that among 899 cases of drug-induced liver injury enrolled in a US prospective study between 2004 and 2013, no cases were attributed to denosumab or other agents used to treat osteoporosis [18]. The only reported case of DILI related to Prolia has been reported by our group at Kaplan Medical Center [7]. In this case, to identify the correlation between denosumab and the patient’s liver toxicity, the authors examined the temporal relationship, the amount of drug administered and the duration of the treatment with denosumab. Exposure to over-the-counter medications and both herbal and dietary supplements was ruled out. A lymphocyte toxicity assay (LTA) was performed to determine if denosumab was involved in the liver injury. LTA is based upon incubating the lymphocytes of the specific individual with the drug that produced the reaction (Denosumab) in the presence or absence of a cytochrome P450 system. After 24 h, the lymphocytes are exposed to the yellow tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The LTA of the patient indicated 31% toxicity upon incubation with the monoclonal antibody vs. control. This result implicated denosumab possibly being responsible for the liver reaction. Primary biliary cholangitis and other chronic liver diseases are associated with osteoporosis. There are reports showing lower levels of RANKL in these diseases than in controls [19]. More than this, circulating mononuclear cells could have a higher capacity to differentiate into osteoclasts in patients with chronic liver disease and osteopenia [20]. Presently, Osteoporosis Canada Clinical Practice Guidelines recommend this drug as a first-line therapy for postmenopausal osteoporosis [21]. Wensel’s group summarized serious denosumab-induced infections, dermatologic adverse reactions, as well as possible hypocalcemia [22]. Adverse effects to denosumab are reported in dentistry. A 74-year-old woman treated with denosumab developed osteonecrosis of the mandible [23]. In a recent review article [24], the authors studied bone biopsies collected from patients exposed to denosumab that presented infectious, inflammatory and necrotic jaw diseases. They found different degrees of bone histological changes. However, none of the individuals presented hepatic disturbances [24]. In our first description of denosumab-induced DILI [7], we demonstrated a possible mechanism by which denosumab could cause severe liver toxicity: by blocking and actively reducing RANKL under normal blood levels. In the first report, we described a 72-year-old woman. The liver biopsy revealed a sub-massive hepatic necrosis consistent with DILI. It was suggested that denosumab might produce a cholestatic reaction. The drug also induced the elevation of members of the TNF family and inflammatory chemokines. Because of the prolonged half-life of denosumab (25.4 days), this immune activation can be a long-lasting event. In the present case, the person is also a woman, but she is younger. It is not clear why the onset of the DILI occurred so long after the initiation of Prolia therapy. This suggests immunological priming and, together with the response to steroids, is consistent with an immune mechanism and not autoimmune hepatitis. There is a single report of the production of denosumab-binding antibodies. Two patients who were treated with 100 mg of denosumab administered every six months developed such antibodies, after a month of treatment in one patient and after 12 months in the other. Upon a bioassay evaluation, these were not found to be neutralizing antibodies. They were not detected in subsequent samples [4]. The long half-life of denosumab may play a role in the development of DILI, with a prolonged exposure at low doses. Denosumab reported worldwide sales of $701 million in the third quarter of 2020. It is imperative to follow up on patients for a longer period of time in order to ensure the safety of this drug. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author Contributions V.O. and S.M. took care of the patient during her hospitalization, S.I.-S., A.Y. and N.Z.S. performed the pathological determinations at Kaplan Medical Center in Rehovot, Israel. M.N. performed all the biochemical and immuno-genetic analyses. She also uses her expertise to provide a pharma-toxicological assessment of the case. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. The laboratory analysis and the medical support was part of the standard of care offered by the hospital. Neuman’s work was funded by In Vitro Drug Safety and Biotechnology. Institutional Review Board Statement The patient was treated during her hospitalization at Kaplan Medical Center in Rehovot, Israel. The biopsy and the blood of the patient was taken during her hospitalization and analyzed in the laboratory. All the procedures were part of her standard of care. As a result ethical review and approval were waived for this study. Informed Consent Statement The patient did not need to sign an informed consent since she received the standard of care in the Kaplan Hospital. She agreed in to the presentation of the case report as a scientific publication. Data Availability Statement Data supporting reported results can be found at the hospital recording database. Conflicts of Interest The authors declare no conflict of interest. All the authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All the authors contributed to the study concept and design: acquisition, analysis, and interpretation of data and drafting of the manuscript and revision of the manuscript for intellectual content. Technical support was provided by In Vitro Drug Safety and Biotechnology. Abbreviations AI aromatase inhibitor ALT alanine aminotransferase (glutamic-pyruvic transaminase, SGPT) ALP alkaline phosphatase ANA antinuclear antibody AST aspartate aminotransferase (glutamic-oxalo-acetic transaminase, SGOT) BMD bone mineral density CK cytokeratin CBC compleate blood count CMV cytomegalo-virus DILI drug-induced liver injury EBV Epstein–Bar virus ER estrogen receptor FASLG FAS Ligand–tumor necrosis factor SF6 (TNF-SF6), CD95L/APO1L FFPE Formalin-fixed paraffin-embedded tissue GGT-γ glutamyl-transferase GnRh gonadotropin releasing hormone HBV hepatitis virus B HCV hepatitis virus C H & E Haematoxylen-Eosin IL Interleukin: IL1-α IL1β, IL2, IL4, IL6, IL8, IL 15 INR International Normalized Ratio LTA lymphocyte toxicity assay MTT 3-(4,5-dimethyl-thiazol-2-yl)-2,5-dio-phenyl tetrazolium bromide NFκB nuclear factor-κB OPC-K osteoclast protease cathepsin K OPG osteo-protegerin RANKL receptor activator of nuclear factor-κB ligand TB Total Bilirubin (summation of conjugated and nonconjugated serum bilirubin) ULN Upper Limit of the Normal reference range Figures and Schemes biomedicines-09-00076-sch001_Scheme 1Scheme 1 ALT and AST kinetics. biomedicines-09-00076-sch002_Scheme 2Scheme 2 Kinetics of γGT. Figure 1 H&E staining, plasma cells infiltrate (see in the circle). Figure 2 H&E staining showing lobular inflammation in the portal area. Figure 3 H&E staining showing portal inflammation & inter-phase hepatitis. Figure 4 Masson Trichome staining showing fibrosis of the portal tracts (right top corner). Figure 5 CytoKeratin 7 (CK7) staining showing bile ductular proliferation in the portal tract (marked by the blue square). Figure 6 Positive MUM-1 of plasma cells in the portal tract.	Subcutaneous	DrugAdministrationRoute	CC BY	33466886	18,819,705	2021-01-14
What was the outcome of reaction 'Drug-induced liver injury'?	Denosumab-Induced Immune Hepatitis. Denosumab-Prolia®, Xgeva® (Amgen) is a fully human antibody to the receptor activator of the nuclear factor-K ligand (RANKL). Hepatotoxicity is extremely rare, with only one reported case of immune origin. We present a second case of hepatotoxicity resulting from an immune reaction to denosumab. A 43-year-old female was referred to the Endocrinology, Diabetes & Metabolism Department for treatment of low bone mineral density (BMD) following endocrine therapy with letrozole and lucrin because of breast cancer. She developed premature menopause at the age of 36 years when she underwent a left lumpectomy due to an infiltrating duct carcinoma of the breast (T1 NO MO) and was subsequently started on endocrine therapy. Denosumab was started to prevent osteoporosis. On the third year after starting on denosumab and one month after she received the last injection, she became ill. The routine biochemical analysis showed that the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) rose appreciatively to 10 times the upper limit of normal (ULN). The gamma-glutamyl transferase (GGT) level was elevated slightly to 67 U/L (0-38 U/L). The serum gamma-globulin level was elevated to 1.72 g/dL (0.7-1.6 gr/dl), while the total bilirubin (TB) and serum albumin levels were normal. A liver biopsy revealed a moderate to severe chronic inflammatory infiltrate containing MUM-1 positive plasma cells. In addition, numerous CD-3 positive small T lymphocytes and few CD-20 positive B lymphocytes and eosinophils were seen in the portal tracts. Moderate to severe interface hepatitis, bile duct proliferation and mild portal fibrosis were also identified. The results could be consistent with the diagnosis of drug-induced liver injury (DILI). 1. Introduction Denosumab is a human monoclonal antibody targeting the key bone resorption mediator receptor activator of the nuclear factor kappa B (NF-κB) ligand (RANKL). Additionally, denosumab contains odanacatib, a specific inhibitor of the osteoclast protease cathepsin K (OPC-K), and antibodies against the two endogenous inhibitors of bone formation, the proteins sclerostin and dickkopf-1. The drug is administered via subcutaneous injection once every six months and is approved for the treatment of postmenopausal women with osteoporosis at an increased risk of fracture [1]. Denosumab is also one of the first-line treatments recommended for aromatase inhibitor-induced bone loss [2,3]. The onset of menopause is associated with a decrease of the estrogen concentration. The function of the receptor activator of the NF-κB ligand, RANKL, is to support the binding of the receptor activator RANK to the osteoclast [4]. Denosumab binding to RANKL inhibits osteoclast formation, maintenance and survival, and reduces bone resorption and turnover. It has a half-life of 25.4 days. Drug-induced liver injury (DILI) occurs as an unpredicted response to therapeutic doses of a medication (an idiosyncratic reaction–Type B) or as a response to a high dose of the medication (intrinsic toxicity–Type A), which are associated with hepatic injury [5,6]. Type A reactions are a direct result of the drugs’ therapeutic effect. The reaction is dose- and frequency-dependent. Type B reactions are unexpected, unrelated to the dose and frequency of administration, and only occur in a minority of patients who are presumed to have a predisposition [5,6]. In this paper, we present an example of denosumab-induced immune hepatitis. DILI encompasses both acute and/or chronic hepatic lesions. The liver injury may be the only clinical manifestation of the adverse drug effect, or it may be accompanied by injury to other organs or by systemic manifestations. This article supports the safe and effective use of drugs by patients and guides laboratory medicine professionals in determining the possible DILI. We report a second rare case of DILI resulting from exposure to denosumab. 2. Case Report A 43-year-old female was referred to our Endocrinology, Diabetes & Metabolism Department for treatment of low bone mineral density (BMD) following endocrine therapy of breast cancer. She developed premature menopause at the age of 36 years, when she underwent a left lumpectomy due to an infiltrating duct carcinoma of the breast (T1 NO MO). She subsequently underwent adjuvant chemo-radiotherapy. The tumor was estrogen receptor (ER) positive. Due to her history of anticardiolipin syndrome, tamoxifen, a selective ER modulator treatment with an increased risk of thrombosis was contraindicated. She commenced treatment with a combination of an aromatase inhibitor (AI), letrozole, and a gonadotropin-releasing hormone (Gn RH agonist), lucrin, for estrogen suppression. The tablet contains 2.5 mg of letrozole, a nonsteroidal aromatase inhibitor. Femara [4,4′-(1H-1,2,4-Triazol-1-ylmethylene)] inhibits estrogen synthesis. There are no reported drug interactions between lucrin, letrazole or denosumab. The dual-energy X-ray absorptiometry scan T score in the lumbar spine was −2.2, and an oral bisphosphonate was started. In the next year, during bisphosphonate treatment, she developed gastrointestinal symptoms resulting in her treatment being changed to subcutaneous denosumab 60 mg every six months. She also took vitamin D and a calcium replacement. There was no past history of fracture. The patient did not smoke or consume alcohol. The only other therapeutic she took was aspirin. On the third year after starting on denosumab and one month after she had received a subcutaneous administration of the drug, a gradual rise of liver enzymes was noted. The patient became tired and reported a poor appetite. The physical examination was unremarkable. Laboratory investigations revealed a complete blood count (CBC) and international normalized ratio (INR) within normal limits. An elevation of serum transaminases (ALT and AST) to around 10 times ULN was observed. Routine virology tests for the determination of hepatitis virus B (HBV) and virus hepatitis C (HCV), as well as Epstein–Bar virus (EBV) and cytomegalovirus (CMV), were negative. The routine autoimmune serology values for smooth muscle antibody, mitochondrial antibody and anti-nuclear antibody (ANA) were negative. The value for soluble liver antigen-antibody (Ag Ab) was 2.3 (0.0–20.0 units), the value for anti proteinase was 3 < 0.2 (0.00–0.99 index), and the value for anti myeloperoxidase was < 0.2 (0.00–0.99 index). The liver kidney microsome Ab-(LKM, liver kidney microsome andibody) was also negative. There was no evidence of chronic hepatitis. The GGT level was 67 U/L (0–38 U/L). The serum gamma-globulin level was 1.72 g/dL (0.7–1.6 gr/dl). The total bilirubin (TB) and serum albumin levels were normal. An abdominal ultrasound was normal. Scheme 1 shows the longitudinal transaminases data in this patient, from the time she started denosumab, during hospitalization and after stopping the treatment. The levels of the γGT follow the same pattern. As shown in Scheme 2, the enzyme levels spike during the liver toxic episode and decline after the interruption of the therapy. The elevation of this enzyme points to a cholestatic process. 3. Pathology-Methods Samples in formalin were embedded in the Pathology Institute into formalin-fixed paraffin-embedded tissue (FFPE) blocks. The blocks were sectioned by Leica EM2245 microtome into 3–5 µm slices, which were loaded on StarFrost microscope slides for H&E staining or on Leica X-tra Adhesive slides for immunohistochemistry (IHC) staining. Hematoxylin and eosin (H&E) staining slides were loaded onto Sakura’s Tissue-Tek© Prisma & Film for the automatic staining and covering of the slide. The IHC staining protocol includes: slides’ incubation at 60 °C for 45 min, xylen incubation for removal of paraffin, hematoxilyn (PRC LTd., 17610, Harris, Jefferson, OR, USA) incubation for 10 min (two washes for 5 min each) incubation in 70% ethanol for washing, 10 min of incubation in Eosin (Pioneer Research Chemical Ltd., 18553, Colchester, UK) staining, two washings × 5 min incubation with 70% ethanol, 2 washings × 5 min incubation in 100% ETOH, three times × 5 min in Xylen and cover slipping with Xylen film. All procedures were done according to the company’s protocol & recommendations. To read the fibrin deposits, Masson trychrome staining was employed. The slides were loaded on Ultra Bench Ventana and stained according to the validated protocol with cytokeratin (CK)7 Antibody (DAKO, Clone: OVTL12/30, Dil: 1:100, UltraView, standard, 32 min Ab incubation). An additional slide was stained with MUM1 Ab (clone: MUM1p; Dil: 1:50; DBS; Ultra View standard, 37 °C, 42′ + Amplifier). Stains were performed with a Ventana Ultra Bench stain apparatus using an ultraView Universal DAB Detection Kit (Catalog Number: 760). 4. Histology A liver biopsy was performed, which revealed a moderate to severe chronic inflammatory infiltrate. Specifically, the liver core biopsy revealed a well-preserved liver architecture. The parenchyma had a few foci of inflammation. In the portal tracts, there was a moderate to severe infiltrate containing plasma cells, numerous positive CD3+ small T lymphocytes, and few C 20 positive B lymphocytes and eosinophils (Figure 1, Figure 2 and Figure 3). Hyperplasia and mild portal fibrosis were identified using Masson staining (Figure 4). Immunostaining revealed a severe interface hepatitis with bile ductular proliferation on CK-7 staining (Figure 5). Immunohistochemistry (Figure 6) presents an infiltrate containing MUM-1 positive plasma cells in the portal tracts. The likely diagnosis was considered to be consistent with immune hepatitis due to DILI. 5. Discussion Osteoporosis. is characterized by low bone mass, microarchitectural disruption and skeletal fragility, resulting in decreased bone strength and increased risk of fracture. Endocrine therapy, an adjuvant treatment in the management of patients with hormone-sensitive breast cancer is associated with adverse effects on the musculoskeletal system and commonly increases the risk of osteoporosis and fractures. Angiogenesis and osteogenesis connect for the bone function. Blood vessels supply oxygen, nutrients and progenitor cells to the osteogenic environment. The functional vasculature system cooperates for physiological bone healing [7]. The identification of novel therapeutic targets to prevent the disease aimed at inhibiting bone resorption and increasing bone formation. In our patient Denosumab was prescribed to treat aromatase induced bone loss. The combination of the clinical illness together with biochemical abnormalities, the temporal association and the lack of any other suspects remains highly suspicious for denosumab-induced toxic events. The calculated score of 6 (a probable drug-induced adverse reaction) was obtained using the Naranjo score [8]. A causality assessment via the updated RUCAM was done [9]. The updated RUCAM takes into account divergent laboratory analyses of liver injury and classifies it into two different subscales: the hepatocellular type of injury and the cholestatic or mixed type of injury. These types can be differentiated using the ratio R, calculated as the ALT/ALP activity measured at the time of liver injury and expressed as a multiple of ULN. In our case, the liver injury was consistent with hepatocellular and cholestatic injury, and the RUCAM score was 4, leading to a possible causality level of DILI from denosumab. It appears unlikely that a direct hepatotoxic effect would appear after three years of use, and thus the possibility of an immune-mediated drug-induced liver injury was considered more likely. Oral steroid treatment with budesonide at 9 mg/day was commenced. Over the course of the following months, there was a slow decline in the levels of the transaminases and the GGT. Currently the liver tests are normalized, and the patient remains in good health on maintenance therapy with steroids. Denosumab is an effective antiresorptive drug; however, discontinuation of the drug can result in an accelerated bone turnover, rapid loss of bone mineral density and an increased rate of multiple vertebral fractures. However, since the hepatic illness may recur upon reintroduction of the offending drug, the rechallenge of denosumab was not considered ethically possible, especially in view of its prolonged half-life. After denosumab was stopped, treatment was switched to intravenous zolendronic acid at 5 mg/year. The last dual-energy X-ray absorptiometry scan T score was −2.1 at the lumbar spine (BMD 0.925g/cm2), −1.6 at the neck of the femur (BMD 0.817 g/cm2) and −0.6 at the forearm (BMD 0.638 g/cm2). We describe a case of a patient following a lumpectomy because of a duct carcinoma of the breast. Since the tumor expressed a hormone receptor, she was started on endocrine therapy. Consistent with recommendations from several international expert groups [2,3,4,5,6,7,8,10] for the assessment of aromatase inhibitor-induced bone loss, an antiresorptive treatment with oral biphosphonate was started. During the follow-up visits, the bisphosphonate therapy was switched to denosumab. On the third year after starting on denosumab, a gradual rise of liver enzymes was noted. The abnormal liver testing and the result of the liver biopsy suggested a diagnosis of DILI. The temporal relation, the prolonged half-life of the drug and the absence of other causes implicate denosumab as the possible cause of hepatotoxicity, so the drug was stopped. We believe that the liver damage was immune-related due to denosumab. The prolonged steroid treatment needed for the liver disease adds another factor with deleterious effects on the musculoskeletal system and increases the risk of osteoporosis and fractures in our patient. For the preservation of bone health, multiple risk factors are taken into consideration. The treatment varies from basic bone protective measures to specific antiresorptive therapy, and the treatment duration should last for as long as the course of the endocrine therapy is given [11]. For this reason, and because the rechallenge of denosumab was not considered ethically possible, it was extremely important to demonstrate the causality of the drug. Since the liver has a dual blood supply, both systemic and portal, many drugs can cause hepatotoxicity, including those affecting the musculo-skeletal system [12]. Menopause results in a decrease in estrogen concentrations and consequently an enhanced osteoclast maturation and activity. In addition, there is a decrease in the osteoblast function. RANKL and the osteoprotegerin (OPG) system have central roles in the the osteoclast activity, RANKL is expressed on both preosteoblast and stromatolite cell surfaces and plays a central role in osteoclastogenesis by binding to the RANK receptor. RANKL plays an important role in osteoclast differentiation and activation and also in the inhibition of osteoclast apoptosis. OPG, secreted by both osteoblasts and stromatolite cells in the bone marrow, is a soluble member of the tumor necrosis factor superfamily that decreases the RANKL activity and thus inhibits osteoclasts’ differentiation, resulting in a decrease in the osteoclast activity [11]. Since Prolia was approved by the FDA in 2010 for the treatment of osteoporosis, it has been implicated in a large number of clinical trials. Denosumab was not associated with changes in serum aminotransferase levels, and the rates of adverse reactions were similar in patients who received denosumab or placebo. A study population has been published in 2006. The authors described 412 postmenopausal women with a low bone density treated with denosumab [three doses every three or six months], versus alendronate or placebo for 12 months. The bone density increased with denosumab and alendronate but not with placebo. The authors did not find changes in the blood chemistry results [4]. Lewieck et al. [13] followed up the 412 women treated with denosumab, alendronate or a placebo. The incidence of side-effects was similar in all three groups and did not change during the second year of the study. There were “no clinically relevant changes in either serum chemistry or hematology values” [13]. Another study of 255 women with breast cancer and bone metastases treated with denosumab for at least 13 weeks found no “unexpected” changes in liver enzymes [14]. Cummings et al. (2009) reported a randomized placebo-controlled trial of 7868 postmenopausal women with osteoporosis treated with denosumab or a placebo [every six months] for three years. Bone minerals increased and there were fewer fractures in the denosumab-treated patients. The adverse events were similar except for eczema [3% vs. 1.7%], cellulitis [0.3% vs. <0.1%] and flatulence [2.2% vs. 1.4%]. The authors did not describe ALT elevations or hepatotoxicity [15]. Bone et al. (2013) followed 3547 postmenopausal women with osteoporosis treated with denosumab for three or six years. The authors declared that the side-effects did not increase with time; they did not note transaminase elevations or DILI [16]. An additional study described no hepatotoxicity in 4550 postmenopausal women with osteoporosis that were treated for another seven years with denosumab. Serious adverse events included osteonecrosis of the jaw (n = 13) and atypical fractures, but there was no mention of ALT elevations. In their systematic review and meta-analysis where the authors described the efficacy and safety of denosumab in the therapy of bone metastases, they did not mention ALT elevations or hepatotoxicity [17]. Moreover, the Drug Induced Liver Injury Network (DILIN) group reported that among 899 cases of drug-induced liver injury enrolled in a US prospective study between 2004 and 2013, no cases were attributed to denosumab or other agents used to treat osteoporosis [18]. The only reported case of DILI related to Prolia has been reported by our group at Kaplan Medical Center [7]. In this case, to identify the correlation between denosumab and the patient’s liver toxicity, the authors examined the temporal relationship, the amount of drug administered and the duration of the treatment with denosumab. Exposure to over-the-counter medications and both herbal and dietary supplements was ruled out. A lymphocyte toxicity assay (LTA) was performed to determine if denosumab was involved in the liver injury. LTA is based upon incubating the lymphocytes of the specific individual with the drug that produced the reaction (Denosumab) in the presence or absence of a cytochrome P450 system. After 24 h, the lymphocytes are exposed to the yellow tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The LTA of the patient indicated 31% toxicity upon incubation with the monoclonal antibody vs. control. This result implicated denosumab possibly being responsible for the liver reaction. Primary biliary cholangitis and other chronic liver diseases are associated with osteoporosis. There are reports showing lower levels of RANKL in these diseases than in controls [19]. More than this, circulating mononuclear cells could have a higher capacity to differentiate into osteoclasts in patients with chronic liver disease and osteopenia [20]. Presently, Osteoporosis Canada Clinical Practice Guidelines recommend this drug as a first-line therapy for postmenopausal osteoporosis [21]. Wensel’s group summarized serious denosumab-induced infections, dermatologic adverse reactions, as well as possible hypocalcemia [22]. Adverse effects to denosumab are reported in dentistry. A 74-year-old woman treated with denosumab developed osteonecrosis of the mandible [23]. In a recent review article [24], the authors studied bone biopsies collected from patients exposed to denosumab that presented infectious, inflammatory and necrotic jaw diseases. They found different degrees of bone histological changes. However, none of the individuals presented hepatic disturbances [24]. In our first description of denosumab-induced DILI [7], we demonstrated a possible mechanism by which denosumab could cause severe liver toxicity: by blocking and actively reducing RANKL under normal blood levels. In the first report, we described a 72-year-old woman. The liver biopsy revealed a sub-massive hepatic necrosis consistent with DILI. It was suggested that denosumab might produce a cholestatic reaction. The drug also induced the elevation of members of the TNF family and inflammatory chemokines. Because of the prolonged half-life of denosumab (25.4 days), this immune activation can be a long-lasting event. In the present case, the person is also a woman, but she is younger. It is not clear why the onset of the DILI occurred so long after the initiation of Prolia therapy. This suggests immunological priming and, together with the response to steroids, is consistent with an immune mechanism and not autoimmune hepatitis. There is a single report of the production of denosumab-binding antibodies. Two patients who were treated with 100 mg of denosumab administered every six months developed such antibodies, after a month of treatment in one patient and after 12 months in the other. Upon a bioassay evaluation, these were not found to be neutralizing antibodies. They were not detected in subsequent samples [4]. The long half-life of denosumab may play a role in the development of DILI, with a prolonged exposure at low doses. Denosumab reported worldwide sales of $701 million in the third quarter of 2020. It is imperative to follow up on patients for a longer period of time in order to ensure the safety of this drug. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author Contributions V.O. and S.M. took care of the patient during her hospitalization, S.I.-S., A.Y. and N.Z.S. performed the pathological determinations at Kaplan Medical Center in Rehovot, Israel. M.N. performed all the biochemical and immuno-genetic analyses. She also uses her expertise to provide a pharma-toxicological assessment of the case. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. The laboratory analysis and the medical support was part of the standard of care offered by the hospital. Neuman’s work was funded by In Vitro Drug Safety and Biotechnology. Institutional Review Board Statement The patient was treated during her hospitalization at Kaplan Medical Center in Rehovot, Israel. The biopsy and the blood of the patient was taken during her hospitalization and analyzed in the laboratory. All the procedures were part of her standard of care. As a result ethical review and approval were waived for this study. Informed Consent Statement The patient did not need to sign an informed consent since she received the standard of care in the Kaplan Hospital. She agreed in to the presentation of the case report as a scientific publication. Data Availability Statement Data supporting reported results can be found at the hospital recording database. Conflicts of Interest The authors declare no conflict of interest. All the authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All the authors contributed to the study concept and design: acquisition, analysis, and interpretation of data and drafting of the manuscript and revision of the manuscript for intellectual content. Technical support was provided by In Vitro Drug Safety and Biotechnology. Abbreviations AI aromatase inhibitor ALT alanine aminotransferase (glutamic-pyruvic transaminase, SGPT) ALP alkaline phosphatase ANA antinuclear antibody AST aspartate aminotransferase (glutamic-oxalo-acetic transaminase, SGOT) BMD bone mineral density CK cytokeratin CBC compleate blood count CMV cytomegalo-virus DILI drug-induced liver injury EBV Epstein–Bar virus ER estrogen receptor FASLG FAS Ligand–tumor necrosis factor SF6 (TNF-SF6), CD95L/APO1L FFPE Formalin-fixed paraffin-embedded tissue GGT-γ glutamyl-transferase GnRh gonadotropin releasing hormone HBV hepatitis virus B HCV hepatitis virus C H & E Haematoxylen-Eosin IL Interleukin: IL1-α IL1β, IL2, IL4, IL6, IL8, IL 15 INR International Normalized Ratio LTA lymphocyte toxicity assay MTT 3-(4,5-dimethyl-thiazol-2-yl)-2,5-dio-phenyl tetrazolium bromide NFκB nuclear factor-κB OPC-K osteoclast protease cathepsin K OPG osteo-protegerin RANKL receptor activator of nuclear factor-κB ligand TB Total Bilirubin (summation of conjugated and nonconjugated serum bilirubin) ULN Upper Limit of the Normal reference range Figures and Schemes biomedicines-09-00076-sch001_Scheme 1Scheme 1 ALT and AST kinetics. biomedicines-09-00076-sch002_Scheme 2Scheme 2 Kinetics of γGT. Figure 1 H&E staining, plasma cells infiltrate (see in the circle). Figure 2 H&E staining showing lobular inflammation in the portal area. Figure 3 H&E staining showing portal inflammation & inter-phase hepatitis. Figure 4 Masson Trichome staining showing fibrosis of the portal tracts (right top corner). Figure 5 CytoKeratin 7 (CK7) staining showing bile ductular proliferation in the portal tract (marked by the blue square). Figure 6 Positive MUM-1 of plasma cells in the portal tract.	Recovered	ReactionOutcome	CC BY	33466886	18,819,705	2021-01-14
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Anaemia'.	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	ASPIRIN, HEPARIN SODIUM	DrugsGivenReaction	CC BY	33468150	18,953,289	2021-01-19
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Atrial fibrillation'.	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	ASPIRIN, HEPARIN SODIUM	DrugsGivenReaction	CC BY	33468150	18,956,379	2021-01-19
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Atrial thrombosis'.	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	ASPIRIN, HEPARIN SODIUM	DrugsGivenReaction	CC BY	33468150	18,956,379	2021-01-19
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Brachiocephalic vein thrombosis'.	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	ASPIRIN, HEPARIN SODIUM	DrugsGivenReaction	CC BY	33468150	18,956,379	2021-01-19
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Cerebral infarction'.	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	ASPIRIN, HEPARIN SODIUM	DrugsGivenReaction	CC BY	33468150	18,956,379	2021-01-19
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Cerebrovascular accident'.	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	ASPIRIN, HEPARIN SODIUM	DrugsGivenReaction	CC BY	33468150	18,953,289	2021-01-19
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Haemoperitoneum'.	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	ASPIRIN, HEPARIN SODIUM	DrugsGivenReaction	CC BY	33468150	18,956,379	2021-01-19
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Heparin-induced thrombocytopenia'.	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	ASPIRIN, HEPARIN SODIUM	DrugsGivenReaction	CC BY	33468150	18,918,196	2021-01-19
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Hypotension'.	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	ASPIRIN, HEPARIN SODIUM	DrugsGivenReaction	CC BY	33468150	18,953,289	2021-01-19
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Paradoxical drug reaction'.	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	ASPIRIN, HEPARIN SODIUM	DrugsGivenReaction	CC BY	33468150	18,953,289	2021-01-19
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Splenic infarction'.	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	ASPIRIN, HEPARIN SODIUM	DrugsGivenReaction	CC BY	33468150	18,956,379	2021-01-19
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Splenic vein thrombosis'.	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	ASPIRIN, HEPARIN SODIUM	DrugsGivenReaction	CC BY	33468150	18,956,379	2021-01-19
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Thrombosis'.	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	ASPIRIN, HEPARIN SODIUM	DrugsGivenReaction	CC BY	33468150	18,918,196	2021-01-19
What is the weight of the patient?	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	66.7 kg.	Weight	CC BY	33468150	18,918,196	2021-01-19
What was the administration route of drug 'HEPARIN SODIUM'?	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	Intravenous bolus	DrugAdministrationRoute	CC BY	33468150	18,918,196	2021-01-19
What was the outcome of reaction 'Anaemia'?	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	Recovered	ReactionOutcome	CC BY	33468150	18,953,289	2021-01-19
What was the outcome of reaction 'Atrial thrombosis'?	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	Recovered	ReactionOutcome	CC BY	33468150	18,956,379	2021-01-19
What was the outcome of reaction 'Brachiocephalic vein thrombosis'?	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	Recovered	ReactionOutcome	CC BY	33468150	18,944,741	2021-01-19
What was the outcome of reaction 'Cerebrovascular accident'?	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	Recovered	ReactionOutcome	CC BY	33468150	18,953,289	2021-01-19
What was the outcome of reaction 'Hypotension'?	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	Recovered	ReactionOutcome	CC BY	33468150	18,953,289	2021-01-19
What was the outcome of reaction 'Paradoxical drug reaction'?	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	Recovered	ReactionOutcome	CC BY	33468150	18,953,289	2021-01-19
What was the outcome of reaction 'Splenic haemorrhage'?	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	Recovered	ReactionOutcome	CC BY	33468150	18,953,289	2021-01-19
What was the outcome of reaction 'Splenic infarction'?	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	Recovered	ReactionOutcome	CC BY	33468150	18,953,289	2021-01-19
What was the outcome of reaction 'Splenic vein thrombosis'?	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	Recovered	ReactionOutcome	CC BY	33468150	18,953,289	2021-01-19
What was the outcome of reaction 'Thrombosis'?	Heparin-induced thrombocytopenia presenting as splenic hemorrhage following cardiac surgery: a case report. BACKGROUND Heparin-induced thrombocytopenia with thrombosis (HITT) is a paradoxical prothrombotic complication of anticoagulant therapy. As many as 3% of patients undergoing cardiac surgery develop clinical HIT presenting as thrombocytopenia with or without thrombosis within 5-10 days of heparin exposure. Thrombotic complications associated with HIT carry a mortality rate of 5-10%. METHODS We report a case of atraumatic splenic hemorrhage due to splenic vein thrombosis as the main indicator of HIT following cardiac surgery in a 62-year-old woman. She presented to the emergency department on day nine following coronary artery bypass graft surgery with acute weakness, dizziness, and malaise. Her evaluation in the emergency department found anemia and thrombocytopenia. A coagulation profile revealed a markedly elevated d-dimer. She underwent a computed tomography scan of the chest, abdomen and pelvis for suspected bleed and was found to have splenic vein thrombosis, right atrial filling defects consistent with atrial thrombus and mild to moderate hemoperitoneum. Surgical consultation was obtained due to splenic hemorrhage. Hematology was consulted on post-operative day 10, however, she unfortunately developed left sided weakness concerning for stroke. A magnetic resonance imaging scan of the brain demonstrated infarct involving distribution of the right anterior cerebral artery. A transesophageal echocardiogram demonstrated a large immobile thrombus within the right atrium with a second, mobile thrombus arising from the left tricuspid valve annulus. Due to a 4Ts score of 7 and markedly positive platelet factor 4 (PF4) IgG antibody a serotonin release assay was not performed given the high probability of HIT. She was cautiously treated with bivalirudin and was transitioned to warfarin anticoagulation. In the following days her platelet count recovered and 3 months later a transthoracic echocardiogram revealed solution of the intracardiac thrombi. CONCLUSIONS Atraumatic splenic hemorrhage is an unusual presentation of HIT that is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal necrosis that also occurs in HIT. Alternative anticoagulation is the mainstay of therapy for HIT despite hemorrhage, given the underlying acquired hypercoagulability. Despite similarities of the presentation between splenic hemorrhage and bilateral adrenal hemorrhage, splenic hemorrhage is rarely described in the literature. HIT should be considered in patients presenting with thrombocytopenia following cardiac surgery. Essentials HIT-associated antibodies are frequently detected after cardiac surgery We report a case of atraumatic splenic hemorrhage due to HIT following coronary artery bypass grafting. HIT should be suspected for any patient with congestive hemorrhage following venous thrombosis, thrombocytopenia, and recent heparin exposure Hemorrhagic splenic infarction due to splenic vein thrombosis is reminiscent of bilateral adrenal hemorrhage (BAH) due to adrenal necrosis seen in HIT Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy which occurs more commonly in postoperative than medical patients [1]. Up to 50% of patients undergoing cardiac surgery develop antibodies to platelet factor 4 (PF4) complexed to heparin, attributed to platelet activation and high heparin doses during cardiopulmonary bypass [2]. Only 1 to 3% of these patients will develop clinical HIT manifesting with thrombocytopenia with or without thrombosis [2, 3]. HIT typically presents 5–10 days following exposure to heparin, often with platelet decline greater than 50% of baseline and is associated with life and limb-threatening thrombotic complications. HIT should be suspected in any patient who has received heparin and develops thrombocytopenia, given high mortality (5–10%) typically a result of thrombotic complications [1]. One increasingly recognized and unusual thrombotic complication that is seen in HIT is adrenal vein thrombosis with tissue necrosis leading to bilateral adrenal hemorrhage (BAH). This congestive hemorrhage following venous thrombosis should raise high clinical suspicion for HIT. We present a case of splenic vein thrombosis post-cardiac surgery complicated hemorrhagic infarct, reminiscent of that seen in BAH, that led to the diagnosis of HIT. Case presentation A 62-year-old woman with a history of congestive heart failure, coronary artery disease, diabetes mellitus type 2, and recent coronary artery bypass graft (CABG) presented to the emergency department with acute weakness, dizziness, and malaise. Past surgery history was significant for a two-vessel coronary artery bypass graft involving the left internal mammary artery to the left anterior descending artery and the left radial to obtuse marginal artery nine days prior to presentation. In the perioperative period she received three heparin boluses at 40u/kg based on a weight of 66.7 kg as well as a heparin infusion dosed between 16u/kg/hr-26u/kg/hr. for 3 days. She had no family history of hematological disorders or hypercoagulability. She was on low-dose aspirin. Her complete blood count (CBC) was normal prior to CABG including platelet count of 268 x 10P3P/μL (normal 150–379 x 10P3P/μL) and at hospital discharge was 174 x10P3P/μL with post-operative hematocrit of 36% (normal 37.5–51%). On physical examination she was afebrile, hypotensive with systolic blood pressure in the 70s, and in rapid atrial fibrillation with heart rate 120–130 s. She denied abdominal pain and had a benign abdominal examination. Laboratory data revealed white blood cell count 10.7 x 10P9/PL (normal 3.4–10.8 x 10P9/PL), hematocrit 19%, and platelet count of 49 x10P3P/μL. Complete metabolic panel and lactate were normal. Troponin was elevated at 0.21 ng/mL (normal 0.00–0.07 ng/mL. Chest x-ray was normal. A limited beside echocardiogram demonstrated no pericardial effusion. She received intravenous fluid resuscitation and transferred to the intensive care unit for further evaluation. Upon arrival to the intensive care unit, she received 2 units of packed red blood cells with appropriate increase to hematocrit 25% post-transfusion and 1 unit of platelets with increase to 73 x10P3P/μL. Further laboratory evaluation was negative for hemolysis with normal lactate dehydrogenase 292 U/L (normal 119–226 U/L) and haptoglobin 98 mg/dL (normal 34–200 mg/dL). A disseminated intravascular coagulopathy (DIC) panel demonstrated d-dimer of > 40.00 μg/ml (normal <=0.49 μg/ml), normal fibrinogen 214 mg/dL (normal 175–475 mg/dL), international normalized ratio (INR) 1.4 (normal 0.9–1.1), protime (PT) 16.7 s (normal 12.0–14.4 s), partial thromboplastin time (PTT 37.3 s (normal 22.0–35.0 s), normal thrombin time (TT), and normal red blood cell morphology. To evaluate for bleeding, a computed tomography (CT) angiogram of the chest, abdomen, and pelvis was ordered and demonstrated filling defects suggestive of right atrial thrombus, splenic vein. thrombosis with splenic infarct, and mild to moderate associated hemoperitoneum. She denied abdominal trauma. Surgery service was consulted and concerned about splenic hemorrhage. Hematology was consulted to evaluate the etiology of spontaneous splenic vein thrombosis with associated hemorrhagic infarction. Unfortunately, on the day of Hematology evaluation (post-operative day 10) she developed left-sided weakness and balance changes concerning for stroke and an MRI revealed infarct involving the distribution of the right anterior cerebral artery. A transesophageal echocardiogram revealed large immobile thrombus in the right atrium with a second thrombus adjacent to this which was mobile and. arising from the left tricuspid valve annulus, large thrombus in the left atrial appendage, and absence of a patent foramen ovale (Fig. 1). She was not a candidate for thrombolytic therapy due to thrombocytopenia. She was calculated to have a 4Ts score of 7. A heparin PF4 IgG antibody was. Fig. 1 Transesophageal Echocardiogram (TEE). White arrow demonstrating immobile right atrial thrombus. Striped arrow demonstrates second mobile thrombus. Grey arrow demonstrates thrombin strands originating from mobile thrombus markedly positive with 3.290 OD. Serotonin release assay was not performed due to the high probability of HIT evidenced by the markedly positive PF4 IgG and 4Ts score of 7. Together these predictive tools indicate a 72% likelihood ratio and 99% posttest probability of HIT [4]. Lower extremity ultrasound was negative for thrombosis while upper extremity ultrasound showed cephalic vein thrombosis. The patient was diagnosed with heparin-induced thrombocytopenia with thrombosis (HITT). Given extensive thrombosis in HIT, anticoagulation was cautiously started with bivalirudin. Our organization utilizes direct thrombin inhibitor (DTI) levels to monitor and adjust bivalrudin dosing. Given the presence of hemoperitoneum prior to bivalruin dosing, bivalrudin was started at 10.01 mg/hr. and titrated to 12.012 mg/hr. based on DTI levels and a dose of 0.13–0.156 mg/kg/hr. at a weight of 77 kg. DTI levels were maintained at goal between 60 and 90 s throughout therapy. She remained clinically stable from the intrabdominal hemorrhage and transitioned to warfarin anticoagulation. Platelets recovered to 108 x10P3P/μL four days after starting alternative anticoagulation and greater than 150 x10P3P/μL nine days after therapy for HITT initiated. The patient had repeat transthoracic echocardiogram after 3 months of warfarin anticoagulation with the resolution of intra-cardiac thrombi. She continues on long-term anticoagulation for atrial fibrillation. Discussion and conclusions Heparin-induced thrombocytopenia (HIT) is a paradoxical prothrombotic syndrome resulting from exposure to heparin-containing products [5–8]. HIT-associated antibodies are frequently detected after cardiac surgery [9]. Thrombosis in the setting of HIT is more commonly associated with the venous system, however, arterial thrombus formation is more common in patients receiving heparin for cardiovascular disease [5]. We report a case of atraumatic splenic hemorrhage due to splenic vein infarction as the main indicator of the diagnosis of HIT. The presentation is reminiscent of the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis that has been reported in HIT. Given the high mortality of HIT, as high as 28% in cardiac surgery patients, awareness of these unique presentations is critical for earlier intervention [10]. HIT should be suspected in any patient who has had prior heparin exposure who presents with thrombocytopenia, especially if the fall is greater than 50% of baseline count and whether the nadir remains within the normal range [6]. Our patient had a high 4 T score and HIT PF4 antibody OD of 3.2 and new splenic and atrial thrombosis, confirming a diagnosis of HITT. Anticoagulation was started with parenteral direct thrombin inhibitor and transitioned to warfarin once platelet count was within the normal limit for 2 consecutive days. Alternative anticoagulation is the mainstay of therapy for HITT even in the setting of hemorrhage, given the underlying acquired hypercoagulability. A well-documented and increasingly well-recognized thrombotic complication of HIT is bilateral adrenal hemorrhage [7]. Clues to this rare complication include the development of abdominal pain, diarrhea, and fever which often lead to imaging of the abdomen and revelation of injury to the adrenal glands and is confirmed by corticotropin-stimulation test [7, 11]. The risk of BAH in a case study of HIT by Warkentin et. al was 1.6% [12]. Strong arterial blood supply with a single central vein makes the. adrenal glands susceptible to congestive hemorrhage following venous thrombosis [13]. We hypothesize that the spleen is at similar risk given it too has a rich arterial supply and single splenic vein. A literature review reveals few reported cases of splenic vein thrombosis with hemorrhagic infarction as a clinical sequela of HIT. Earlier reports discussed spontaneous splenic ruptures occurring on heparin anticoagulants, however did not report on HIT antibody testing [14–17]. One case report had a very similar presentation of splenic hematoma after cardiac surgery, with splenectomy pathology showing multiple fibrin thrombi in the spleen [9]. This pathology confirms our hypothesis of microthrombi leading to subsequent hemorrhagic conversion in the spleen, as occurs in BAH. Early imaging to identify these unusual intra-abdominal thrombotic complications of HIT is encouraged [18]. A case report by Lammering et al. discusses a case of splenic rupture secondary to splenic vein thrombosis resulting from HIT, while the discussion focuses on the utility of multidetector-. row computed tomography in the rapid and accurate diagnosis of HIT related thromboembolic complication. Lammering et al. describe the case in the setting of non-specific abdominal pain which differed from our patient without abdominal pain [19]. Treatment of HIT involves alternative non-heparin anticoagulation in accordance with published guidelines including those by the American College of Chest Physicians and the updated American Society of Hematology Guidelines for Management of Venous Thromboembolism: Heparin-Induced Thrombocytopenia [1, 20]. Despite hemorrhagic complications such as BAH, or as in our patient intraabdominal hemorrhage due to splenic infarction, treatment is careful anticoagulation given the underlying etiology for hemorrhage is hypercoagulability. Multiple cases reports on BAH advocate for prompt initiation of appropriate anticoagulation to ensure a successful outcome [12, 21]. We advocate for a similar approach to congestive splenic hemorrhage following splenic vein thrombosis as a consequence of HIT. This case emphasizes that the diagnosis of HIT should be considered for patients with unusual intra-abdominal thrombosis or hemorrhage, especially within the expected time course following heparin exposure. Congestive hemorrhage following venous thrombosis, with recent heparin exposure and declining platelet count, should raise clinical suspicion of HIT. The diagnosis of HIT-induced splenic vein thrombosis and subsequent hemorrhagic infarct mimics the rare bilateral adrenal hemorrhage due to adrenal vein thrombosis seen in HIT. Multidetector-row computed tomography should be considered for the evaluation of thromboembolic sequelae in patients with unexplained anemia or abdominal pain with suspicion of HITT to exclude adrenal or splenic involvement. Alternative anticoagulation remains the mainstay of therapy. Abbreviations BAHbilateral adrenal hemorrhage CABGcoronary artery bypass graft CBCcomplete blood count CTcomputed tomography DICdisseminated intravascular coagulopathy HITTheparin induced thrombocytopenia with thrombosis HITheparin induced thrombocytopenia PTprotime PTTpartial thromboplastin time TTthrombin time Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. None. Authors’ contributions JF wrote the manuscript. JF, SY, PW LH treated the patient. LH revised and edited the manuscript. All authors have read and approved the manuscript. Funding None. Availability of data and materials No datasets were developed or analyzed for this case report. Ethics approval and consent to participate In the United States, a case report does not require ethics approval. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests.	Recovered	ReactionOutcome	CC BY	33468150	18,918,196	2021-01-19
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Chloroma'.	Case Report: Myeloid Sarcoma Development During Treatment for B Cell Lymphoblastic Lymphoma in a Boy with KRAS/NRAS Gene Mutations. Here, we report a rare case of a 12-year-old boy who was initially diagnosed with B cell lymphoblastic lymphoma (BLBL) and developed myeloid sarcoma (MS) eight months after chemotherapy. Next-generation sequencing (NGS) showed mutations of KRAS and NRAS genes in both the bone marrow and lymph node. He presented an abnormal karyotype of 46, XY, -9, der (16) t (9; 16) (q13; q12), +mar. He received chemotherapy according to the South China Children's Leukemia Group 2016 protocol. Complete remission was achieved by the 15th day post-treatment. Eight months later and immediately prior to the start of maintenance therapy, the patient developed fever, skin nodules in both upper arms, and enlargement of bilateral testes. Pathological analysis of skin and testicular biopsies suggested the diagnosis of myeloid sarcoma (MS). Again, NGS examination showed mutations of KRAS and NRAS genes. The patient underwent haploidentical hematopoietic stem cell transplantation but unfortunately did not survive. The interval of eight-month interval between the initial disease onset and MS brings into question whether MS developed as part of the initial onset of disease or as a secondary tumor in association with chemotherapy. Thus, understanding the pathogenesis of MS involving abnormalities of lymphoid progenitors may assist in the prediction of prognosis and development of novel target therapies. Background Myeloid sarcoma (MS) is characterized as a myeloid neoplasm that mainly affects extramedullary sites, including the skin, lymph nodes, gastrointestinal tract, bone, soft tissue, and reproductive organs.1 Although MS affects all age groups of patients, the incidence of MS is low. MS is most often found in patients with newly or recently diagnosed acute myeloid leukemia (AML). The concurrence of MS with neoplasms derived from lymphoid progenitors has only been reported in few cases.2,3 Mutations of genes related to intracellular signaling pathways (such as the RAS/RAF/MAPK and phosphoinositide-3 pathways) may alter the cell cycle via abnormal proliferation, differentiation, and apoptosis of cells.4 Among these, alterations of genes involved in the receptor tyrosine kinase (RTK)-RAS pathway, including NRAS and KRAS, are suspected to contribute significantly to the regulation and pathogenesis of MS.5,6 Patients who develop MS may have a poor prognosis. Therapies aimed at these genetic variations may help with the development of targeted strategies against MS. Case Presentation A 12-year-old male patient with complaints of neck masses for a month and intermittent fever for two weeks was admitted to the Department of Hematology and Oncology at Shenzhen Children’s Hospital, China. He was diagnosed with lymphadenitis at a local hospital a month before admission and was treated with intravenous ceftriaxone and oral amoxicillin, as well as intravenous methylprednisolone (1.5mg/kg/day) for two days. The patient was previously healthy and denied any family history of malignant diseases. Physical examination revealed multiple enlarged cervical and inguinal lymph nodes. The lymph nodes were moderate to firm in texture, with a maximum size of 2 cm x 1 cm, and mobile without adhesions to each other or neighboring tissues. The lymph nodes were slightly tender on palpation. Hepatosplenomegaly was noted. No other abnormalities were observed during the physical examination. Laboratory evaluations revealed slight leukopenia with a white blood cell count of 4.37 x 109/L and a neutrophil count of 2.15 x 109/L. Neither anemia nor thrombocytopenia was observed. The blood smear examination showed that blasts accounted for 5% of total white blood cells. Elevated levels of alanine aminotransferase (ALT, 170 IU/L), aspartate aminotransferase (AST, 175 IU/L), and lactic dehydrogenase (LDH, 1007 IU/L) were observed. All other laboratory findings (including direct and indirect bilirubin, creatinine, albumin, creatine kinase, and immunoglobulin levels) were within the normal range. Test results for antibodies to tuberculosis, Epstein-Barr virus, cytomegalovirus, Mycoplasma pneumoniae, and Chlamydia pneumoniae were negative. Enhanced computed tomography (CT) scans of the neck and chest confirmed the presence of multiple enlarged cervical lymph nodes, particularly on the left side of the neck. No fluctuations or adhesions of the lymph nodes were observed. Hepatomegaly (3 cm below the lowest right costal margin) and splenomegaly (2.4 cm below the lowest left costal margin) were observed on ultrasound examination. No masses were observed in the abdomen or pelvis. Brain magnetic resonance imaging showed no abnormalities. The bone marrow smear showed 15% of blast cells. These cells displayed a monotonous population of round and oval cells with scant cytoplasm. Vacuolated cytoplasm was observed in a few blasts. Approximately 1% of the blast cells showed basophilic staining with scant cytoplasm, folded nuclei, and absent nucleoli. Periodic acid-Schiff (PAS) staining was positive in most blasts, and peroxidase (POX) staining was positive in 43% of blast cells (Figure 1).Figure 1 Morphological examination of the bone marrow showed cells that displayed a monotonous population of round and oval cells with scant cytoplasm by Giemsa staining. Peroxidase (POX) staining was positive in 43% of blast cells. Periodic acid-Schiff (PAS) staining was positive in most blasts. Flow cytometry analysis of bone marrow aspirates showed blasts accounting for 17.1% of the total cells. Blast cells were positive to CD19, CD10, HLA-DR, TdT, CD13 (partial), CD79a (partial), and myeloperoxidase (MPO; partial) (Figure 2). Expressions of CD34, CD33, CD117, CD20, CD15, CD3, CD56, and IgM were negative. Cytogenetic abnormalities with a karyotype of 46, XY, −9, der (16) t (9; 16) (q13; q12), +mar were observed (Figure 3). Examinations of fusion genes for both acute lymphoblastic leukemia and myeloid leukemia were negative. Panel sequencing was performed to examine genes that related to lymphoid and myeloid neoplasms. Mutations of KRAS (c.38G>A, p.G13D) and NRAS (c.35G>A, p.G12D) genes were detected from the bone marrow sample. The variant allele fractions were 8.3% and 11.1%, respectively.Figure 2 Flow cytometry plots of the bone marrow at the time of diagnosis showed blasts (CD45−) that stained positive for CD10, CD19, CD79a, TdT, and MPO, and negative for CD34, CD20, IgM. Figure 3 Cytogenetic examination of the bone marrow aspirates showed abnormal karyotype of 46, XY, −9, der (16) t (9; 16) (q13; q12), +mar. Analysis of chromosomes showed a deletion of chromosome 9 (red box), translocation of chromosome 9 and 16 (arrow) that formed a derivative chromosome (circle). A biopsy of the cervical lymph nodes on the left side of the neck was performed. Morphological examination showed uniformly small to intermediate-sized cells. Immunohistochemistry (IHC) examination of the lymph node showed positive staining of CD10, CD79a, TdT, Ki-67 (>85%), PAX5, CD1a (partial), CD20 (sporadic), CD3 (isolated), CD4 (few, isolated), CD7 (sporadic), CD8 (sporadic), CD99/MIC2, and CD2 (sporadic). MPO expression was observed (Figure 4). Expressions of ALK, CD30, and CD34 were negative. Next-generation sequencing (NGS) examination of the lymph node showed a mutation of the NRAS gene (c.35G>A, p.G12D). However, mutation of the KRAS gene was not detected in the lymph node. B cell receptor (BCR) rearrangement examination was positive. According to the 2016 WHO classification, a diagnosis of BLBL was considered despite the expression of MPO.7 This patient was categorized as an intermediate-risk patient and was given chemotherapy according to the South China Children’s Leukemia Group 2016 (SCCLG-2016) protocol.Figure 4 Histological examination of the cervical lymph node biopsy of the left neck showed uniformly small to intermediate-sized cells by hematoxylin and eosin (H & E) staining. Immunohistochemistry examination showed strong positive staining for CD10, CD79a, TdT, Ki-67 (>85%), PAX5, and myeloperoxidase, and partial staining of CD20. Chemotherapy consisted of a prednisone experiment for seven days prior to induction therapy, an induction therapy of a cycle of VDLD (VCR/Dex/L-Peg-Asp/DNR) and two cycles of CAM+VL (CTX/Ara-C/6-MP+VCR/Preg-L-Asp), a consolidation therapy of four cycles of high dose MTX, and a reinduction therapy of a cycle of VDLD and a cycle of CAM+VL. Maintenance chemotherapy was prescribed with 6-MP+MTX+VD (VCR+Dex) and planned for a total period of 2.5 years. The patient achieved a good response to prednisone with a significant reduction of blasts in the peripheral blood on the 8th day of therapy. Bone marrow examination performed on the 15th day showed complete remission (CR) of the bone marrow by morphological examination. Flow cytometry of the bone marrow aspirate showed that the minimal residue disease was less than 0.01%. The bone marrow of the patient remained in CR during the time of chemotherapy. No life-threatening infections or complications were observed. Eight months after the initiation of chemotherapy and just prior to the start of maintenance therapy, the patient developed fever, skin nodules in both upper arms, and bilateral testicular enlargements. Both morphological and flow cytometry examination of the bone marrow performed at this time were negative. Biopsies of the skin and testes were performed. Histological examination of testicular tissues showed massive infiltration of tumor cells. IHC examination of the cells revealed positive staining for lysozyme, CD33, MPO, and Ki-67 (>95+), and negative staining for CD7, PAX-5, CD34, TdT. Histological examination of skin tissue showed massive proliferation of tumor cells. IHC of these cells revealed positive staining for lysozyme, MPO, CD33, Ki-67 (>95%+), CD4 (mild), and negative staining for CD3, CD7, CD20, PAX-5, CD117, CD163, CD123, TdT, CD30, ALK. These results suggested MS of the skin and testes. NGS examinations of the skin and testicular samples showed mutations of both the KRAS (c.436G>A, p.A146T) and NRAS genes (c.35G>A, p.G12D). Positron emission CT examination showed nodular lesions with increased uptake of 18F-fluorodeoxyglucose (18F-FDG) in both testes. In addition, increased 18F-FDG uptakes were observed in the cervical vertebra, thoracic vertebra, and the right back ribs, suggesting metastasis to the bones. The patient was subsequently given high-intensity chemotherapy consisting of etoposide, Ara-C, and DNR. After two cycles of chemotherapy, he received haploidentical hematopoietic stem cell transplantation. His mother served as the donor. Unfortunately, this patient did not survive due to severe graft-versus-host disease and other complications following the transplant. Discussion MS is commonly seen in conjunction with, or during relapses of AML, myeloproliferative neoplasm, and myelodysplastic syndrome. MS may be observed in up to 40% of children with AML.1,8,9 However, the incidence of MS remains low within the general oncology pediatric population. MS complicated with acute lymphoblastic leukemia or lymphoma only has been identified rare cases,2,3,10,11 most of which were initially misdiagnosed as non-Hodgkin’s lymphoma. Thus, extensive evaluations were required to overcome the challenges in diagnosing MS complicated with other diseases, especially among those without bone marrow involvement. In the present case, the key presenting abnormalities of the patient were the enlarged cervical and inguinal lymph nodes. Masses were not observed at other sites. Blasts comprised 20% of the total bone marrow cells and expressed markers for B cell blasts, including CD19, CD79a, and PAX5. The patient’s lymph node biopsy results also featured typical histological characteristics of lymphoblastic lymphoma, including the expression of CD10, CD79a, and TdT. An unusual finding of this case was the strong reaction of MPO, a typical myeloid marker identified in the lymph node by immunohistochemistry. MPO was also detected at low level in the blasts by flow cytometry. Another pan-myeloid marker, CD13, was partially demonstrated in the bone marrow by flow cytometry (not shown). However, this is a pan-myeloid marker commonly seen in B-ALL, especially for B-ALL that involves the translocation of chromosomes 9 and 22 and fusion of BCR/ABL1 genes.12,13 Other myeloid associated markers including CD15 and CD117 were not expressed. Expression of monocytic markers including CD14 and CD64 was not observed. In fact, MPO expression could be observed in up to 22% of pediatric BALL patients.14 Another concern in the diagnosis of this patient was the use of methylprednisolone before admission that might have lysed part of the blasts. Thus, the percentage of blasts in the bone marrow (15%) could have been underestimated hence suggestive of a diagnosis of mixed phenotypic acute leukemia (MPAL) according to the 2016 WHO guideline. However, based on the existing evaluations of the bone marrow and lymph nodes, a diagnosis of BLBL with MPO expression was considered for this patient. The cause of MS in this patient remains uncertain. An important aspect of this case was the mutation of both the NRAS and KRAS genes. Concurrent mutations of both NRAS and KRAS gene have rarely been reported because these mutations are often mutually exclusive. This is because additional mutations of the RAS genes would not escalate the discomposed RAS signaling pathway.15 However, the presence of both NRAS and KRAS mutations has been previously identified in few pediatric BALL patients.16 In this case, both NRAS and KRAS mutations were detected at initial diagnosis and at the development of MS. Moreover, NRAS and KRAS genes participate as part of the receptor tyrosine kinase (RTK)-RAS pathway.5,17 Recent genomic studies reveal that mutations associated with the RTK-RAS pathway are frequently observed in pediatric patients with B-ALL and are probably more prevalent in the Chinese population.15,16,18 Although mutations of RAS genes have been shown associated with poorer outcomes in pediatric ALL, AML, MPAL, and infant leukemia patients, the association of RAS mutations and patient prognosis remains undefined, warranting larger cohort studies.15,19 In this case, MS was identified approximately eight months after the initiation of chemotherapy. A plausible explanation is that MS developing at extramedullary sites requires more oncogenetic mutations.9 The high-intensity chemotherapy given to this patient may have accelerated the mutations that led to the development of MS as a second tumor.20–24 This patient had the abnormal chromosomal karyotype of 46, XY, −9, der (16) t (9; 16) (q13; q12), +mar. However, no evidence suggests that this chromosomal abnormality contributes to an increased susceptibility to oncogenic clonal evolution. Whether these chromosomal abnormalities increased the risk for the development of secondary tumors requires further investigation. Another hypothesis of the cause of MS was the cross-lineage transdifferentiation of B cell progenitors in BLBL to MS. In adults, transdifferentiations of BALL or other lymphoid progenitors into neoplasms of other lineage such as histiocytic sarcoma/leukemia have been reported in few cases.25–28 Transdifferentiations between neoplasms of tumor cells from other lineages have also been reported in sporadic adult cases.29,30 Most of these patients feature chromosomal and genetic abnormalities including the mutation of RAS genes. The presence of NRAS and KRAS genes and the expression of MPO may suggest the potential of transdifferentiation of B cells into myeloid blasts. However, transdifferentiation of BLBL to MS has not been reported in pediatric patients and needs further investigations. Conclusions In conclusion, we report a boy who developed MS after chemotherapy for BLBL after a period of approximately eight months. The exact mechanism of the development of MS in this patient remains uncertain. A diagnosis of BLBL was considered despite the expression of myeloid marker MPO. This patient exhibited certain cytogenetic abnormalities, but fusion genes were not presented. NGS evaluations revealed mutations of both NRAS and KRAS genes that may contribute to the genetic origin of MS. Therefore, understanding the mechanisms of the development of MS in this patient may help with the prediction of prognosis and the creation and advancement of therapeutic strategies for targeted treatments, and prevention of secondary malignancies. Acknowledgments We thank A/Prof Ronald Grant from the Hospital for Sick Children in Toronto, Canada for the long-time support to help us to provide better care for our pediatric oncology patients. Xiuli Yuan and Uet Yu are co-first authors for this study. Abbreviations 6-MP, 6-mercaptopurine; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; Ara-C, cytosine arabinoside; BLBL, B cell lymphoblastic lymphoma; CTX, cyclophosphamide; Dex, dexamethasone; DNR, daunoblastina; L-Peg-Asp, L-asparaginase; MPO, myeloperoxidase; MS, myeloid sarcoma; MTX, methotrexate; VCR, vincristine. Data Sharing Statement Not applicable. Ethical Approval and Informed Consent Ethical approval is not required for this Case report. Written informed consent was obtained from the patient’s parent. Consent for Publication Written informed consent was obtained from the patient’s parent for publication of this paper and any accompanying images. Author Contributions All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. Disclosure The authors have no competing interests to declare.	ASPARAGINASE, CYCLOPHOSPHAMIDE, CYTARABINE, DAUNORUBICIN, DEXAMETHASONE, MERCAPTOPURINE, METHOTREXATE, PREDNISONE, VINCRISTINE	DrugsGivenReaction	CC BY-NC	33469311	19,436,569	2021
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Second primary malignancy'.	Case Report: Myeloid Sarcoma Development During Treatment for B Cell Lymphoblastic Lymphoma in a Boy with KRAS/NRAS Gene Mutations. Here, we report a rare case of a 12-year-old boy who was initially diagnosed with B cell lymphoblastic lymphoma (BLBL) and developed myeloid sarcoma (MS) eight months after chemotherapy. Next-generation sequencing (NGS) showed mutations of KRAS and NRAS genes in both the bone marrow and lymph node. He presented an abnormal karyotype of 46, XY, -9, der (16) t (9; 16) (q13; q12), +mar. He received chemotherapy according to the South China Children's Leukemia Group 2016 protocol. Complete remission was achieved by the 15th day post-treatment. Eight months later and immediately prior to the start of maintenance therapy, the patient developed fever, skin nodules in both upper arms, and enlargement of bilateral testes. Pathological analysis of skin and testicular biopsies suggested the diagnosis of myeloid sarcoma (MS). Again, NGS examination showed mutations of KRAS and NRAS genes. The patient underwent haploidentical hematopoietic stem cell transplantation but unfortunately did not survive. The interval of eight-month interval between the initial disease onset and MS brings into question whether MS developed as part of the initial onset of disease or as a secondary tumor in association with chemotherapy. Thus, understanding the pathogenesis of MS involving abnormalities of lymphoid progenitors may assist in the prediction of prognosis and development of novel target therapies. Background Myeloid sarcoma (MS) is characterized as a myeloid neoplasm that mainly affects extramedullary sites, including the skin, lymph nodes, gastrointestinal tract, bone, soft tissue, and reproductive organs.1 Although MS affects all age groups of patients, the incidence of MS is low. MS is most often found in patients with newly or recently diagnosed acute myeloid leukemia (AML). The concurrence of MS with neoplasms derived from lymphoid progenitors has only been reported in few cases.2,3 Mutations of genes related to intracellular signaling pathways (such as the RAS/RAF/MAPK and phosphoinositide-3 pathways) may alter the cell cycle via abnormal proliferation, differentiation, and apoptosis of cells.4 Among these, alterations of genes involved in the receptor tyrosine kinase (RTK)-RAS pathway, including NRAS and KRAS, are suspected to contribute significantly to the regulation and pathogenesis of MS.5,6 Patients who develop MS may have a poor prognosis. Therapies aimed at these genetic variations may help with the development of targeted strategies against MS. Case Presentation A 12-year-old male patient with complaints of neck masses for a month and intermittent fever for two weeks was admitted to the Department of Hematology and Oncology at Shenzhen Children’s Hospital, China. He was diagnosed with lymphadenitis at a local hospital a month before admission and was treated with intravenous ceftriaxone and oral amoxicillin, as well as intravenous methylprednisolone (1.5mg/kg/day) for two days. The patient was previously healthy and denied any family history of malignant diseases. Physical examination revealed multiple enlarged cervical and inguinal lymph nodes. The lymph nodes were moderate to firm in texture, with a maximum size of 2 cm x 1 cm, and mobile without adhesions to each other or neighboring tissues. The lymph nodes were slightly tender on palpation. Hepatosplenomegaly was noted. No other abnormalities were observed during the physical examination. Laboratory evaluations revealed slight leukopenia with a white blood cell count of 4.37 x 109/L and a neutrophil count of 2.15 x 109/L. Neither anemia nor thrombocytopenia was observed. The blood smear examination showed that blasts accounted for 5% of total white blood cells. Elevated levels of alanine aminotransferase (ALT, 170 IU/L), aspartate aminotransferase (AST, 175 IU/L), and lactic dehydrogenase (LDH, 1007 IU/L) were observed. All other laboratory findings (including direct and indirect bilirubin, creatinine, albumin, creatine kinase, and immunoglobulin levels) were within the normal range. Test results for antibodies to tuberculosis, Epstein-Barr virus, cytomegalovirus, Mycoplasma pneumoniae, and Chlamydia pneumoniae were negative. Enhanced computed tomography (CT) scans of the neck and chest confirmed the presence of multiple enlarged cervical lymph nodes, particularly on the left side of the neck. No fluctuations or adhesions of the lymph nodes were observed. Hepatomegaly (3 cm below the lowest right costal margin) and splenomegaly (2.4 cm below the lowest left costal margin) were observed on ultrasound examination. No masses were observed in the abdomen or pelvis. Brain magnetic resonance imaging showed no abnormalities. The bone marrow smear showed 15% of blast cells. These cells displayed a monotonous population of round and oval cells with scant cytoplasm. Vacuolated cytoplasm was observed in a few blasts. Approximately 1% of the blast cells showed basophilic staining with scant cytoplasm, folded nuclei, and absent nucleoli. Periodic acid-Schiff (PAS) staining was positive in most blasts, and peroxidase (POX) staining was positive in 43% of blast cells (Figure 1).Figure 1 Morphological examination of the bone marrow showed cells that displayed a monotonous population of round and oval cells with scant cytoplasm by Giemsa staining. Peroxidase (POX) staining was positive in 43% of blast cells. Periodic acid-Schiff (PAS) staining was positive in most blasts. Flow cytometry analysis of bone marrow aspirates showed blasts accounting for 17.1% of the total cells. Blast cells were positive to CD19, CD10, HLA-DR, TdT, CD13 (partial), CD79a (partial), and myeloperoxidase (MPO; partial) (Figure 2). Expressions of CD34, CD33, CD117, CD20, CD15, CD3, CD56, and IgM were negative. Cytogenetic abnormalities with a karyotype of 46, XY, −9, der (16) t (9; 16) (q13; q12), +mar were observed (Figure 3). Examinations of fusion genes for both acute lymphoblastic leukemia and myeloid leukemia were negative. Panel sequencing was performed to examine genes that related to lymphoid and myeloid neoplasms. Mutations of KRAS (c.38G>A, p.G13D) and NRAS (c.35G>A, p.G12D) genes were detected from the bone marrow sample. The variant allele fractions were 8.3% and 11.1%, respectively.Figure 2 Flow cytometry plots of the bone marrow at the time of diagnosis showed blasts (CD45−) that stained positive for CD10, CD19, CD79a, TdT, and MPO, and negative for CD34, CD20, IgM. Figure 3 Cytogenetic examination of the bone marrow aspirates showed abnormal karyotype of 46, XY, −9, der (16) t (9; 16) (q13; q12), +mar. Analysis of chromosomes showed a deletion of chromosome 9 (red box), translocation of chromosome 9 and 16 (arrow) that formed a derivative chromosome (circle). A biopsy of the cervical lymph nodes on the left side of the neck was performed. Morphological examination showed uniformly small to intermediate-sized cells. Immunohistochemistry (IHC) examination of the lymph node showed positive staining of CD10, CD79a, TdT, Ki-67 (>85%), PAX5, CD1a (partial), CD20 (sporadic), CD3 (isolated), CD4 (few, isolated), CD7 (sporadic), CD8 (sporadic), CD99/MIC2, and CD2 (sporadic). MPO expression was observed (Figure 4). Expressions of ALK, CD30, and CD34 were negative. Next-generation sequencing (NGS) examination of the lymph node showed a mutation of the NRAS gene (c.35G>A, p.G12D). However, mutation of the KRAS gene was not detected in the lymph node. B cell receptor (BCR) rearrangement examination was positive. According to the 2016 WHO classification, a diagnosis of BLBL was considered despite the expression of MPO.7 This patient was categorized as an intermediate-risk patient and was given chemotherapy according to the South China Children’s Leukemia Group 2016 (SCCLG-2016) protocol.Figure 4 Histological examination of the cervical lymph node biopsy of the left neck showed uniformly small to intermediate-sized cells by hematoxylin and eosin (H & E) staining. Immunohistochemistry examination showed strong positive staining for CD10, CD79a, TdT, Ki-67 (>85%), PAX5, and myeloperoxidase, and partial staining of CD20. Chemotherapy consisted of a prednisone experiment for seven days prior to induction therapy, an induction therapy of a cycle of VDLD (VCR/Dex/L-Peg-Asp/DNR) and two cycles of CAM+VL (CTX/Ara-C/6-MP+VCR/Preg-L-Asp), a consolidation therapy of four cycles of high dose MTX, and a reinduction therapy of a cycle of VDLD and a cycle of CAM+VL. Maintenance chemotherapy was prescribed with 6-MP+MTX+VD (VCR+Dex) and planned for a total period of 2.5 years. The patient achieved a good response to prednisone with a significant reduction of blasts in the peripheral blood on the 8th day of therapy. Bone marrow examination performed on the 15th day showed complete remission (CR) of the bone marrow by morphological examination. Flow cytometry of the bone marrow aspirate showed that the minimal residue disease was less than 0.01%. The bone marrow of the patient remained in CR during the time of chemotherapy. No life-threatening infections or complications were observed. Eight months after the initiation of chemotherapy and just prior to the start of maintenance therapy, the patient developed fever, skin nodules in both upper arms, and bilateral testicular enlargements. Both morphological and flow cytometry examination of the bone marrow performed at this time were negative. Biopsies of the skin and testes were performed. Histological examination of testicular tissues showed massive infiltration of tumor cells. IHC examination of the cells revealed positive staining for lysozyme, CD33, MPO, and Ki-67 (>95+), and negative staining for CD7, PAX-5, CD34, TdT. Histological examination of skin tissue showed massive proliferation of tumor cells. IHC of these cells revealed positive staining for lysozyme, MPO, CD33, Ki-67 (>95%+), CD4 (mild), and negative staining for CD3, CD7, CD20, PAX-5, CD117, CD163, CD123, TdT, CD30, ALK. These results suggested MS of the skin and testes. NGS examinations of the skin and testicular samples showed mutations of both the KRAS (c.436G>A, p.A146T) and NRAS genes (c.35G>A, p.G12D). Positron emission CT examination showed nodular lesions with increased uptake of 18F-fluorodeoxyglucose (18F-FDG) in both testes. In addition, increased 18F-FDG uptakes were observed in the cervical vertebra, thoracic vertebra, and the right back ribs, suggesting metastasis to the bones. The patient was subsequently given high-intensity chemotherapy consisting of etoposide, Ara-C, and DNR. After two cycles of chemotherapy, he received haploidentical hematopoietic stem cell transplantation. His mother served as the donor. Unfortunately, this patient did not survive due to severe graft-versus-host disease and other complications following the transplant. Discussion MS is commonly seen in conjunction with, or during relapses of AML, myeloproliferative neoplasm, and myelodysplastic syndrome. MS may be observed in up to 40% of children with AML.1,8,9 However, the incidence of MS remains low within the general oncology pediatric population. MS complicated with acute lymphoblastic leukemia or lymphoma only has been identified rare cases,2,3,10,11 most of which were initially misdiagnosed as non-Hodgkin’s lymphoma. Thus, extensive evaluations were required to overcome the challenges in diagnosing MS complicated with other diseases, especially among those without bone marrow involvement. In the present case, the key presenting abnormalities of the patient were the enlarged cervical and inguinal lymph nodes. Masses were not observed at other sites. Blasts comprised 20% of the total bone marrow cells and expressed markers for B cell blasts, including CD19, CD79a, and PAX5. The patient’s lymph node biopsy results also featured typical histological characteristics of lymphoblastic lymphoma, including the expression of CD10, CD79a, and TdT. An unusual finding of this case was the strong reaction of MPO, a typical myeloid marker identified in the lymph node by immunohistochemistry. MPO was also detected at low level in the blasts by flow cytometry. Another pan-myeloid marker, CD13, was partially demonstrated in the bone marrow by flow cytometry (not shown). However, this is a pan-myeloid marker commonly seen in B-ALL, especially for B-ALL that involves the translocation of chromosomes 9 and 22 and fusion of BCR/ABL1 genes.12,13 Other myeloid associated markers including CD15 and CD117 were not expressed. Expression of monocytic markers including CD14 and CD64 was not observed. In fact, MPO expression could be observed in up to 22% of pediatric BALL patients.14 Another concern in the diagnosis of this patient was the use of methylprednisolone before admission that might have lysed part of the blasts. Thus, the percentage of blasts in the bone marrow (15%) could have been underestimated hence suggestive of a diagnosis of mixed phenotypic acute leukemia (MPAL) according to the 2016 WHO guideline. However, based on the existing evaluations of the bone marrow and lymph nodes, a diagnosis of BLBL with MPO expression was considered for this patient. The cause of MS in this patient remains uncertain. An important aspect of this case was the mutation of both the NRAS and KRAS genes. Concurrent mutations of both NRAS and KRAS gene have rarely been reported because these mutations are often mutually exclusive. This is because additional mutations of the RAS genes would not escalate the discomposed RAS signaling pathway.15 However, the presence of both NRAS and KRAS mutations has been previously identified in few pediatric BALL patients.16 In this case, both NRAS and KRAS mutations were detected at initial diagnosis and at the development of MS. Moreover, NRAS and KRAS genes participate as part of the receptor tyrosine kinase (RTK)-RAS pathway.5,17 Recent genomic studies reveal that mutations associated with the RTK-RAS pathway are frequently observed in pediatric patients with B-ALL and are probably more prevalent in the Chinese population.15,16,18 Although mutations of RAS genes have been shown associated with poorer outcomes in pediatric ALL, AML, MPAL, and infant leukemia patients, the association of RAS mutations and patient prognosis remains undefined, warranting larger cohort studies.15,19 In this case, MS was identified approximately eight months after the initiation of chemotherapy. A plausible explanation is that MS developing at extramedullary sites requires more oncogenetic mutations.9 The high-intensity chemotherapy given to this patient may have accelerated the mutations that led to the development of MS as a second tumor.20–24 This patient had the abnormal chromosomal karyotype of 46, XY, −9, der (16) t (9; 16) (q13; q12), +mar. However, no evidence suggests that this chromosomal abnormality contributes to an increased susceptibility to oncogenic clonal evolution. Whether these chromosomal abnormalities increased the risk for the development of secondary tumors requires further investigation. Another hypothesis of the cause of MS was the cross-lineage transdifferentiation of B cell progenitors in BLBL to MS. In adults, transdifferentiations of BALL or other lymphoid progenitors into neoplasms of other lineage such as histiocytic sarcoma/leukemia have been reported in few cases.25–28 Transdifferentiations between neoplasms of tumor cells from other lineages have also been reported in sporadic adult cases.29,30 Most of these patients feature chromosomal and genetic abnormalities including the mutation of RAS genes. The presence of NRAS and KRAS genes and the expression of MPO may suggest the potential of transdifferentiation of B cells into myeloid blasts. However, transdifferentiation of BLBL to MS has not been reported in pediatric patients and needs further investigations. Conclusions In conclusion, we report a boy who developed MS after chemotherapy for BLBL after a period of approximately eight months. The exact mechanism of the development of MS in this patient remains uncertain. A diagnosis of BLBL was considered despite the expression of myeloid marker MPO. This patient exhibited certain cytogenetic abnormalities, but fusion genes were not presented. NGS evaluations revealed mutations of both NRAS and KRAS genes that may contribute to the genetic origin of MS. Therefore, understanding the mechanisms of the development of MS in this patient may help with the prediction of prognosis and the creation and advancement of therapeutic strategies for targeted treatments, and prevention of secondary malignancies. Acknowledgments We thank A/Prof Ronald Grant from the Hospital for Sick Children in Toronto, Canada for the long-time support to help us to provide better care for our pediatric oncology patients. Xiuli Yuan and Uet Yu are co-first authors for this study. Abbreviations 6-MP, 6-mercaptopurine; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; Ara-C, cytosine arabinoside; BLBL, B cell lymphoblastic lymphoma; CTX, cyclophosphamide; Dex, dexamethasone; DNR, daunoblastina; L-Peg-Asp, L-asparaginase; MPO, myeloperoxidase; MS, myeloid sarcoma; MTX, methotrexate; VCR, vincristine. Data Sharing Statement Not applicable. Ethical Approval and Informed Consent Ethical approval is not required for this Case report. Written informed consent was obtained from the patient’s parent. Consent for Publication Written informed consent was obtained from the patient’s parent for publication of this paper and any accompanying images. Author Contributions All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. Disclosure The authors have no competing interests to declare.	ASPARAGINASE, CYCLOPHOSPHAMIDE, CYTARABINE, DAUNORUBICIN, DEXAMETHASONE, MERCAPTOPURINE, METHOTREXATE, PREDNISONE, VINCRISTINE	DrugsGivenReaction	CC BY-NC	33469311	19,436,569	2021
What was the dosage of drug 'METHOTREXATE'?	Case Report: Myeloid Sarcoma Development During Treatment for B Cell Lymphoblastic Lymphoma in a Boy with KRAS/NRAS Gene Mutations. Here, we report a rare case of a 12-year-old boy who was initially diagnosed with B cell lymphoblastic lymphoma (BLBL) and developed myeloid sarcoma (MS) eight months after chemotherapy. Next-generation sequencing (NGS) showed mutations of KRAS and NRAS genes in both the bone marrow and lymph node. He presented an abnormal karyotype of 46, XY, -9, der (16) t (9; 16) (q13; q12), +mar. He received chemotherapy according to the South China Children's Leukemia Group 2016 protocol. Complete remission was achieved by the 15th day post-treatment. Eight months later and immediately prior to the start of maintenance therapy, the patient developed fever, skin nodules in both upper arms, and enlargement of bilateral testes. Pathological analysis of skin and testicular biopsies suggested the diagnosis of myeloid sarcoma (MS). Again, NGS examination showed mutations of KRAS and NRAS genes. The patient underwent haploidentical hematopoietic stem cell transplantation but unfortunately did not survive. The interval of eight-month interval between the initial disease onset and MS brings into question whether MS developed as part of the initial onset of disease or as a secondary tumor in association with chemotherapy. Thus, understanding the pathogenesis of MS involving abnormalities of lymphoid progenitors may assist in the prediction of prognosis and development of novel target therapies. Background Myeloid sarcoma (MS) is characterized as a myeloid neoplasm that mainly affects extramedullary sites, including the skin, lymph nodes, gastrointestinal tract, bone, soft tissue, and reproductive organs.1 Although MS affects all age groups of patients, the incidence of MS is low. MS is most often found in patients with newly or recently diagnosed acute myeloid leukemia (AML). The concurrence of MS with neoplasms derived from lymphoid progenitors has only been reported in few cases.2,3 Mutations of genes related to intracellular signaling pathways (such as the RAS/RAF/MAPK and phosphoinositide-3 pathways) may alter the cell cycle via abnormal proliferation, differentiation, and apoptosis of cells.4 Among these, alterations of genes involved in the receptor tyrosine kinase (RTK)-RAS pathway, including NRAS and KRAS, are suspected to contribute significantly to the regulation and pathogenesis of MS.5,6 Patients who develop MS may have a poor prognosis. Therapies aimed at these genetic variations may help with the development of targeted strategies against MS. Case Presentation A 12-year-old male patient with complaints of neck masses for a month and intermittent fever for two weeks was admitted to the Department of Hematology and Oncology at Shenzhen Children’s Hospital, China. He was diagnosed with lymphadenitis at a local hospital a month before admission and was treated with intravenous ceftriaxone and oral amoxicillin, as well as intravenous methylprednisolone (1.5mg/kg/day) for two days. The patient was previously healthy and denied any family history of malignant diseases. Physical examination revealed multiple enlarged cervical and inguinal lymph nodes. The lymph nodes were moderate to firm in texture, with a maximum size of 2 cm x 1 cm, and mobile without adhesions to each other or neighboring tissues. The lymph nodes were slightly tender on palpation. Hepatosplenomegaly was noted. No other abnormalities were observed during the physical examination. Laboratory evaluations revealed slight leukopenia with a white blood cell count of 4.37 x 109/L and a neutrophil count of 2.15 x 109/L. Neither anemia nor thrombocytopenia was observed. The blood smear examination showed that blasts accounted for 5% of total white blood cells. Elevated levels of alanine aminotransferase (ALT, 170 IU/L), aspartate aminotransferase (AST, 175 IU/L), and lactic dehydrogenase (LDH, 1007 IU/L) were observed. All other laboratory findings (including direct and indirect bilirubin, creatinine, albumin, creatine kinase, and immunoglobulin levels) were within the normal range. Test results for antibodies to tuberculosis, Epstein-Barr virus, cytomegalovirus, Mycoplasma pneumoniae, and Chlamydia pneumoniae were negative. Enhanced computed tomography (CT) scans of the neck and chest confirmed the presence of multiple enlarged cervical lymph nodes, particularly on the left side of the neck. No fluctuations or adhesions of the lymph nodes were observed. Hepatomegaly (3 cm below the lowest right costal margin) and splenomegaly (2.4 cm below the lowest left costal margin) were observed on ultrasound examination. No masses were observed in the abdomen or pelvis. Brain magnetic resonance imaging showed no abnormalities. The bone marrow smear showed 15% of blast cells. These cells displayed a monotonous population of round and oval cells with scant cytoplasm. Vacuolated cytoplasm was observed in a few blasts. Approximately 1% of the blast cells showed basophilic staining with scant cytoplasm, folded nuclei, and absent nucleoli. Periodic acid-Schiff (PAS) staining was positive in most blasts, and peroxidase (POX) staining was positive in 43% of blast cells (Figure 1).Figure 1 Morphological examination of the bone marrow showed cells that displayed a monotonous population of round and oval cells with scant cytoplasm by Giemsa staining. Peroxidase (POX) staining was positive in 43% of blast cells. Periodic acid-Schiff (PAS) staining was positive in most blasts. Flow cytometry analysis of bone marrow aspirates showed blasts accounting for 17.1% of the total cells. Blast cells were positive to CD19, CD10, HLA-DR, TdT, CD13 (partial), CD79a (partial), and myeloperoxidase (MPO; partial) (Figure 2). Expressions of CD34, CD33, CD117, CD20, CD15, CD3, CD56, and IgM were negative. Cytogenetic abnormalities with a karyotype of 46, XY, −9, der (16) t (9; 16) (q13; q12), +mar were observed (Figure 3). Examinations of fusion genes for both acute lymphoblastic leukemia and myeloid leukemia were negative. Panel sequencing was performed to examine genes that related to lymphoid and myeloid neoplasms. Mutations of KRAS (c.38G>A, p.G13D) and NRAS (c.35G>A, p.G12D) genes were detected from the bone marrow sample. The variant allele fractions were 8.3% and 11.1%, respectively.Figure 2 Flow cytometry plots of the bone marrow at the time of diagnosis showed blasts (CD45−) that stained positive for CD10, CD19, CD79a, TdT, and MPO, and negative for CD34, CD20, IgM. Figure 3 Cytogenetic examination of the bone marrow aspirates showed abnormal karyotype of 46, XY, −9, der (16) t (9; 16) (q13; q12), +mar. Analysis of chromosomes showed a deletion of chromosome 9 (red box), translocation of chromosome 9 and 16 (arrow) that formed a derivative chromosome (circle). A biopsy of the cervical lymph nodes on the left side of the neck was performed. Morphological examination showed uniformly small to intermediate-sized cells. Immunohistochemistry (IHC) examination of the lymph node showed positive staining of CD10, CD79a, TdT, Ki-67 (>85%), PAX5, CD1a (partial), CD20 (sporadic), CD3 (isolated), CD4 (few, isolated), CD7 (sporadic), CD8 (sporadic), CD99/MIC2, and CD2 (sporadic). MPO expression was observed (Figure 4). Expressions of ALK, CD30, and CD34 were negative. Next-generation sequencing (NGS) examination of the lymph node showed a mutation of the NRAS gene (c.35G>A, p.G12D). However, mutation of the KRAS gene was not detected in the lymph node. B cell receptor (BCR) rearrangement examination was positive. According to the 2016 WHO classification, a diagnosis of BLBL was considered despite the expression of MPO.7 This patient was categorized as an intermediate-risk patient and was given chemotherapy according to the South China Children’s Leukemia Group 2016 (SCCLG-2016) protocol.Figure 4 Histological examination of the cervical lymph node biopsy of the left neck showed uniformly small to intermediate-sized cells by hematoxylin and eosin (H & E) staining. Immunohistochemistry examination showed strong positive staining for CD10, CD79a, TdT, Ki-67 (>85%), PAX5, and myeloperoxidase, and partial staining of CD20. Chemotherapy consisted of a prednisone experiment for seven days prior to induction therapy, an induction therapy of a cycle of VDLD (VCR/Dex/L-Peg-Asp/DNR) and two cycles of CAM+VL (CTX/Ara-C/6-MP+VCR/Preg-L-Asp), a consolidation therapy of four cycles of high dose MTX, and a reinduction therapy of a cycle of VDLD and a cycle of CAM+VL. Maintenance chemotherapy was prescribed with 6-MP+MTX+VD (VCR+Dex) and planned for a total period of 2.5 years. The patient achieved a good response to prednisone with a significant reduction of blasts in the peripheral blood on the 8th day of therapy. Bone marrow examination performed on the 15th day showed complete remission (CR) of the bone marrow by morphological examination. Flow cytometry of the bone marrow aspirate showed that the minimal residue disease was less than 0.01%. The bone marrow of the patient remained in CR during the time of chemotherapy. No life-threatening infections or complications were observed. Eight months after the initiation of chemotherapy and just prior to the start of maintenance therapy, the patient developed fever, skin nodules in both upper arms, and bilateral testicular enlargements. Both morphological and flow cytometry examination of the bone marrow performed at this time were negative. Biopsies of the skin and testes were performed. Histological examination of testicular tissues showed massive infiltration of tumor cells. IHC examination of the cells revealed positive staining for lysozyme, CD33, MPO, and Ki-67 (>95+), and negative staining for CD7, PAX-5, CD34, TdT. Histological examination of skin tissue showed massive proliferation of tumor cells. IHC of these cells revealed positive staining for lysozyme, MPO, CD33, Ki-67 (>95%+), CD4 (mild), and negative staining for CD3, CD7, CD20, PAX-5, CD117, CD163, CD123, TdT, CD30, ALK. These results suggested MS of the skin and testes. NGS examinations of the skin and testicular samples showed mutations of both the KRAS (c.436G>A, p.A146T) and NRAS genes (c.35G>A, p.G12D). Positron emission CT examination showed nodular lesions with increased uptake of 18F-fluorodeoxyglucose (18F-FDG) in both testes. In addition, increased 18F-FDG uptakes were observed in the cervical vertebra, thoracic vertebra, and the right back ribs, suggesting metastasis to the bones. The patient was subsequently given high-intensity chemotherapy consisting of etoposide, Ara-C, and DNR. After two cycles of chemotherapy, he received haploidentical hematopoietic stem cell transplantation. His mother served as the donor. Unfortunately, this patient did not survive due to severe graft-versus-host disease and other complications following the transplant. Discussion MS is commonly seen in conjunction with, or during relapses of AML, myeloproliferative neoplasm, and myelodysplastic syndrome. MS may be observed in up to 40% of children with AML.1,8,9 However, the incidence of MS remains low within the general oncology pediatric population. MS complicated with acute lymphoblastic leukemia or lymphoma only has been identified rare cases,2,3,10,11 most of which were initially misdiagnosed as non-Hodgkin’s lymphoma. Thus, extensive evaluations were required to overcome the challenges in diagnosing MS complicated with other diseases, especially among those without bone marrow involvement. In the present case, the key presenting abnormalities of the patient were the enlarged cervical and inguinal lymph nodes. Masses were not observed at other sites. Blasts comprised 20% of the total bone marrow cells and expressed markers for B cell blasts, including CD19, CD79a, and PAX5. The patient’s lymph node biopsy results also featured typical histological characteristics of lymphoblastic lymphoma, including the expression of CD10, CD79a, and TdT. An unusual finding of this case was the strong reaction of MPO, a typical myeloid marker identified in the lymph node by immunohistochemistry. MPO was also detected at low level in the blasts by flow cytometry. Another pan-myeloid marker, CD13, was partially demonstrated in the bone marrow by flow cytometry (not shown). However, this is a pan-myeloid marker commonly seen in B-ALL, especially for B-ALL that involves the translocation of chromosomes 9 and 22 and fusion of BCR/ABL1 genes.12,13 Other myeloid associated markers including CD15 and CD117 were not expressed. Expression of monocytic markers including CD14 and CD64 was not observed. In fact, MPO expression could be observed in up to 22% of pediatric BALL patients.14 Another concern in the diagnosis of this patient was the use of methylprednisolone before admission that might have lysed part of the blasts. Thus, the percentage of blasts in the bone marrow (15%) could have been underestimated hence suggestive of a diagnosis of mixed phenotypic acute leukemia (MPAL) according to the 2016 WHO guideline. However, based on the existing evaluations of the bone marrow and lymph nodes, a diagnosis of BLBL with MPO expression was considered for this patient. The cause of MS in this patient remains uncertain. An important aspect of this case was the mutation of both the NRAS and KRAS genes. Concurrent mutations of both NRAS and KRAS gene have rarely been reported because these mutations are often mutually exclusive. This is because additional mutations of the RAS genes would not escalate the discomposed RAS signaling pathway.15 However, the presence of both NRAS and KRAS mutations has been previously identified in few pediatric BALL patients.16 In this case, both NRAS and KRAS mutations were detected at initial diagnosis and at the development of MS. Moreover, NRAS and KRAS genes participate as part of the receptor tyrosine kinase (RTK)-RAS pathway.5,17 Recent genomic studies reveal that mutations associated with the RTK-RAS pathway are frequently observed in pediatric patients with B-ALL and are probably more prevalent in the Chinese population.15,16,18 Although mutations of RAS genes have been shown associated with poorer outcomes in pediatric ALL, AML, MPAL, and infant leukemia patients, the association of RAS mutations and patient prognosis remains undefined, warranting larger cohort studies.15,19 In this case, MS was identified approximately eight months after the initiation of chemotherapy. A plausible explanation is that MS developing at extramedullary sites requires more oncogenetic mutations.9 The high-intensity chemotherapy given to this patient may have accelerated the mutations that led to the development of MS as a second tumor.20–24 This patient had the abnormal chromosomal karyotype of 46, XY, −9, der (16) t (9; 16) (q13; q12), +mar. However, no evidence suggests that this chromosomal abnormality contributes to an increased susceptibility to oncogenic clonal evolution. Whether these chromosomal abnormalities increased the risk for the development of secondary tumors requires further investigation. Another hypothesis of the cause of MS was the cross-lineage transdifferentiation of B cell progenitors in BLBL to MS. In adults, transdifferentiations of BALL or other lymphoid progenitors into neoplasms of other lineage such as histiocytic sarcoma/leukemia have been reported in few cases.25–28 Transdifferentiations between neoplasms of tumor cells from other lineages have also been reported in sporadic adult cases.29,30 Most of these patients feature chromosomal and genetic abnormalities including the mutation of RAS genes. The presence of NRAS and KRAS genes and the expression of MPO may suggest the potential of transdifferentiation of B cells into myeloid blasts. However, transdifferentiation of BLBL to MS has not been reported in pediatric patients and needs further investigations. Conclusions In conclusion, we report a boy who developed MS after chemotherapy for BLBL after a period of approximately eight months. The exact mechanism of the development of MS in this patient remains uncertain. A diagnosis of BLBL was considered despite the expression of myeloid marker MPO. This patient exhibited certain cytogenetic abnormalities, but fusion genes were not presented. NGS evaluations revealed mutations of both NRAS and KRAS genes that may contribute to the genetic origin of MS. Therefore, understanding the mechanisms of the development of MS in this patient may help with the prediction of prognosis and the creation and advancement of therapeutic strategies for targeted treatments, and prevention of secondary malignancies. Acknowledgments We thank A/Prof Ronald Grant from the Hospital for Sick Children in Toronto, Canada for the long-time support to help us to provide better care for our pediatric oncology patients. Xiuli Yuan and Uet Yu are co-first authors for this study. Abbreviations 6-MP, 6-mercaptopurine; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; Ara-C, cytosine arabinoside; BLBL, B cell lymphoblastic lymphoma; CTX, cyclophosphamide; Dex, dexamethasone; DNR, daunoblastina; L-Peg-Asp, L-asparaginase; MPO, myeloperoxidase; MS, myeloid sarcoma; MTX, methotrexate; VCR, vincristine. Data Sharing Statement Not applicable. Ethical Approval and Informed Consent Ethical approval is not required for this Case report. Written informed consent was obtained from the patient’s parent. Consent for Publication Written informed consent was obtained from the patient’s parent for publication of this paper and any accompanying images. Author Contributions All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. Disclosure The authors have no competing interests to declare.	HIGH?DOSE (4 CYCLIC)	DrugDosageText	CC BY-NC	33469311	19,436,569	2021
What was the dosage of drug 'PREDNISONE'?	Case Report: Myeloid Sarcoma Development During Treatment for B Cell Lymphoblastic Lymphoma in a Boy with KRAS/NRAS Gene Mutations. Here, we report a rare case of a 12-year-old boy who was initially diagnosed with B cell lymphoblastic lymphoma (BLBL) and developed myeloid sarcoma (MS) eight months after chemotherapy. Next-generation sequencing (NGS) showed mutations of KRAS and NRAS genes in both the bone marrow and lymph node. He presented an abnormal karyotype of 46, XY, -9, der (16) t (9; 16) (q13; q12), +mar. He received chemotherapy according to the South China Children's Leukemia Group 2016 protocol. Complete remission was achieved by the 15th day post-treatment. Eight months later and immediately prior to the start of maintenance therapy, the patient developed fever, skin nodules in both upper arms, and enlargement of bilateral testes. Pathological analysis of skin and testicular biopsies suggested the diagnosis of myeloid sarcoma (MS). Again, NGS examination showed mutations of KRAS and NRAS genes. The patient underwent haploidentical hematopoietic stem cell transplantation but unfortunately did not survive. The interval of eight-month interval between the initial disease onset and MS brings into question whether MS developed as part of the initial onset of disease or as a secondary tumor in association with chemotherapy. Thus, understanding the pathogenesis of MS involving abnormalities of lymphoid progenitors may assist in the prediction of prognosis and development of novel target therapies. Background Myeloid sarcoma (MS) is characterized as a myeloid neoplasm that mainly affects extramedullary sites, including the skin, lymph nodes, gastrointestinal tract, bone, soft tissue, and reproductive organs.1 Although MS affects all age groups of patients, the incidence of MS is low. MS is most often found in patients with newly or recently diagnosed acute myeloid leukemia (AML). The concurrence of MS with neoplasms derived from lymphoid progenitors has only been reported in few cases.2,3 Mutations of genes related to intracellular signaling pathways (such as the RAS/RAF/MAPK and phosphoinositide-3 pathways) may alter the cell cycle via abnormal proliferation, differentiation, and apoptosis of cells.4 Among these, alterations of genes involved in the receptor tyrosine kinase (RTK)-RAS pathway, including NRAS and KRAS, are suspected to contribute significantly to the regulation and pathogenesis of MS.5,6 Patients who develop MS may have a poor prognosis. Therapies aimed at these genetic variations may help with the development of targeted strategies against MS. Case Presentation A 12-year-old male patient with complaints of neck masses for a month and intermittent fever for two weeks was admitted to the Department of Hematology and Oncology at Shenzhen Children’s Hospital, China. He was diagnosed with lymphadenitis at a local hospital a month before admission and was treated with intravenous ceftriaxone and oral amoxicillin, as well as intravenous methylprednisolone (1.5mg/kg/day) for two days. The patient was previously healthy and denied any family history of malignant diseases. Physical examination revealed multiple enlarged cervical and inguinal lymph nodes. The lymph nodes were moderate to firm in texture, with a maximum size of 2 cm x 1 cm, and mobile without adhesions to each other or neighboring tissues. The lymph nodes were slightly tender on palpation. Hepatosplenomegaly was noted. No other abnormalities were observed during the physical examination. Laboratory evaluations revealed slight leukopenia with a white blood cell count of 4.37 x 109/L and a neutrophil count of 2.15 x 109/L. Neither anemia nor thrombocytopenia was observed. The blood smear examination showed that blasts accounted for 5% of total white blood cells. Elevated levels of alanine aminotransferase (ALT, 170 IU/L), aspartate aminotransferase (AST, 175 IU/L), and lactic dehydrogenase (LDH, 1007 IU/L) were observed. All other laboratory findings (including direct and indirect bilirubin, creatinine, albumin, creatine kinase, and immunoglobulin levels) were within the normal range. Test results for antibodies to tuberculosis, Epstein-Barr virus, cytomegalovirus, Mycoplasma pneumoniae, and Chlamydia pneumoniae were negative. Enhanced computed tomography (CT) scans of the neck and chest confirmed the presence of multiple enlarged cervical lymph nodes, particularly on the left side of the neck. No fluctuations or adhesions of the lymph nodes were observed. Hepatomegaly (3 cm below the lowest right costal margin) and splenomegaly (2.4 cm below the lowest left costal margin) were observed on ultrasound examination. No masses were observed in the abdomen or pelvis. Brain magnetic resonance imaging showed no abnormalities. The bone marrow smear showed 15% of blast cells. These cells displayed a monotonous population of round and oval cells with scant cytoplasm. Vacuolated cytoplasm was observed in a few blasts. Approximately 1% of the blast cells showed basophilic staining with scant cytoplasm, folded nuclei, and absent nucleoli. Periodic acid-Schiff (PAS) staining was positive in most blasts, and peroxidase (POX) staining was positive in 43% of blast cells (Figure 1).Figure 1 Morphological examination of the bone marrow showed cells that displayed a monotonous population of round and oval cells with scant cytoplasm by Giemsa staining. Peroxidase (POX) staining was positive in 43% of blast cells. Periodic acid-Schiff (PAS) staining was positive in most blasts. Flow cytometry analysis of bone marrow aspirates showed blasts accounting for 17.1% of the total cells. Blast cells were positive to CD19, CD10, HLA-DR, TdT, CD13 (partial), CD79a (partial), and myeloperoxidase (MPO; partial) (Figure 2). Expressions of CD34, CD33, CD117, CD20, CD15, CD3, CD56, and IgM were negative. Cytogenetic abnormalities with a karyotype of 46, XY, −9, der (16) t (9; 16) (q13; q12), +mar were observed (Figure 3). Examinations of fusion genes for both acute lymphoblastic leukemia and myeloid leukemia were negative. Panel sequencing was performed to examine genes that related to lymphoid and myeloid neoplasms. Mutations of KRAS (c.38G>A, p.G13D) and NRAS (c.35G>A, p.G12D) genes were detected from the bone marrow sample. The variant allele fractions were 8.3% and 11.1%, respectively.Figure 2 Flow cytometry plots of the bone marrow at the time of diagnosis showed blasts (CD45−) that stained positive for CD10, CD19, CD79a, TdT, and MPO, and negative for CD34, CD20, IgM. Figure 3 Cytogenetic examination of the bone marrow aspirates showed abnormal karyotype of 46, XY, −9, der (16) t (9; 16) (q13; q12), +mar. Analysis of chromosomes showed a deletion of chromosome 9 (red box), translocation of chromosome 9 and 16 (arrow) that formed a derivative chromosome (circle). A biopsy of the cervical lymph nodes on the left side of the neck was performed. Morphological examination showed uniformly small to intermediate-sized cells. Immunohistochemistry (IHC) examination of the lymph node showed positive staining of CD10, CD79a, TdT, Ki-67 (>85%), PAX5, CD1a (partial), CD20 (sporadic), CD3 (isolated), CD4 (few, isolated), CD7 (sporadic), CD8 (sporadic), CD99/MIC2, and CD2 (sporadic). MPO expression was observed (Figure 4). Expressions of ALK, CD30, and CD34 were negative. Next-generation sequencing (NGS) examination of the lymph node showed a mutation of the NRAS gene (c.35G>A, p.G12D). However, mutation of the KRAS gene was not detected in the lymph node. B cell receptor (BCR) rearrangement examination was positive. According to the 2016 WHO classification, a diagnosis of BLBL was considered despite the expression of MPO.7 This patient was categorized as an intermediate-risk patient and was given chemotherapy according to the South China Children’s Leukemia Group 2016 (SCCLG-2016) protocol.Figure 4 Histological examination of the cervical lymph node biopsy of the left neck showed uniformly small to intermediate-sized cells by hematoxylin and eosin (H & E) staining. Immunohistochemistry examination showed strong positive staining for CD10, CD79a, TdT, Ki-67 (>85%), PAX5, and myeloperoxidase, and partial staining of CD20. Chemotherapy consisted of a prednisone experiment for seven days prior to induction therapy, an induction therapy of a cycle of VDLD (VCR/Dex/L-Peg-Asp/DNR) and two cycles of CAM+VL (CTX/Ara-C/6-MP+VCR/Preg-L-Asp), a consolidation therapy of four cycles of high dose MTX, and a reinduction therapy of a cycle of VDLD and a cycle of CAM+VL. Maintenance chemotherapy was prescribed with 6-MP+MTX+VD (VCR+Dex) and planned for a total period of 2.5 years. The patient achieved a good response to prednisone with a significant reduction of blasts in the peripheral blood on the 8th day of therapy. Bone marrow examination performed on the 15th day showed complete remission (CR) of the bone marrow by morphological examination. Flow cytometry of the bone marrow aspirate showed that the minimal residue disease was less than 0.01%. The bone marrow of the patient remained in CR during the time of chemotherapy. No life-threatening infections or complications were observed. Eight months after the initiation of chemotherapy and just prior to the start of maintenance therapy, the patient developed fever, skin nodules in both upper arms, and bilateral testicular enlargements. Both morphological and flow cytometry examination of the bone marrow performed at this time were negative. Biopsies of the skin and testes were performed. Histological examination of testicular tissues showed massive infiltration of tumor cells. IHC examination of the cells revealed positive staining for lysozyme, CD33, MPO, and Ki-67 (>95+), and negative staining for CD7, PAX-5, CD34, TdT. Histological examination of skin tissue showed massive proliferation of tumor cells. IHC of these cells revealed positive staining for lysozyme, MPO, CD33, Ki-67 (>95%+), CD4 (mild), and negative staining for CD3, CD7, CD20, PAX-5, CD117, CD163, CD123, TdT, CD30, ALK. These results suggested MS of the skin and testes. NGS examinations of the skin and testicular samples showed mutations of both the KRAS (c.436G>A, p.A146T) and NRAS genes (c.35G>A, p.G12D). Positron emission CT examination showed nodular lesions with increased uptake of 18F-fluorodeoxyglucose (18F-FDG) in both testes. In addition, increased 18F-FDG uptakes were observed in the cervical vertebra, thoracic vertebra, and the right back ribs, suggesting metastasis to the bones. The patient was subsequently given high-intensity chemotherapy consisting of etoposide, Ara-C, and DNR. After two cycles of chemotherapy, he received haploidentical hematopoietic stem cell transplantation. His mother served as the donor. Unfortunately, this patient did not survive due to severe graft-versus-host disease and other complications following the transplant. Discussion MS is commonly seen in conjunction with, or during relapses of AML, myeloproliferative neoplasm, and myelodysplastic syndrome. MS may be observed in up to 40% of children with AML.1,8,9 However, the incidence of MS remains low within the general oncology pediatric population. MS complicated with acute lymphoblastic leukemia or lymphoma only has been identified rare cases,2,3,10,11 most of which were initially misdiagnosed as non-Hodgkin’s lymphoma. Thus, extensive evaluations were required to overcome the challenges in diagnosing MS complicated with other diseases, especially among those without bone marrow involvement. In the present case, the key presenting abnormalities of the patient were the enlarged cervical and inguinal lymph nodes. Masses were not observed at other sites. Blasts comprised 20% of the total bone marrow cells and expressed markers for B cell blasts, including CD19, CD79a, and PAX5. The patient’s lymph node biopsy results also featured typical histological characteristics of lymphoblastic lymphoma, including the expression of CD10, CD79a, and TdT. An unusual finding of this case was the strong reaction of MPO, a typical myeloid marker identified in the lymph node by immunohistochemistry. MPO was also detected at low level in the blasts by flow cytometry. Another pan-myeloid marker, CD13, was partially demonstrated in the bone marrow by flow cytometry (not shown). However, this is a pan-myeloid marker commonly seen in B-ALL, especially for B-ALL that involves the translocation of chromosomes 9 and 22 and fusion of BCR/ABL1 genes.12,13 Other myeloid associated markers including CD15 and CD117 were not expressed. Expression of monocytic markers including CD14 and CD64 was not observed. In fact, MPO expression could be observed in up to 22% of pediatric BALL patients.14 Another concern in the diagnosis of this patient was the use of methylprednisolone before admission that might have lysed part of the blasts. Thus, the percentage of blasts in the bone marrow (15%) could have been underestimated hence suggestive of a diagnosis of mixed phenotypic acute leukemia (MPAL) according to the 2016 WHO guideline. However, based on the existing evaluations of the bone marrow and lymph nodes, a diagnosis of BLBL with MPO expression was considered for this patient. The cause of MS in this patient remains uncertain. An important aspect of this case was the mutation of both the NRAS and KRAS genes. Concurrent mutations of both NRAS and KRAS gene have rarely been reported because these mutations are often mutually exclusive. This is because additional mutations of the RAS genes would not escalate the discomposed RAS signaling pathway.15 However, the presence of both NRAS and KRAS mutations has been previously identified in few pediatric BALL patients.16 In this case, both NRAS and KRAS mutations were detected at initial diagnosis and at the development of MS. Moreover, NRAS and KRAS genes participate as part of the receptor tyrosine kinase (RTK)-RAS pathway.5,17 Recent genomic studies reveal that mutations associated with the RTK-RAS pathway are frequently observed in pediatric patients with B-ALL and are probably more prevalent in the Chinese population.15,16,18 Although mutations of RAS genes have been shown associated with poorer outcomes in pediatric ALL, AML, MPAL, and infant leukemia patients, the association of RAS mutations and patient prognosis remains undefined, warranting larger cohort studies.15,19 In this case, MS was identified approximately eight months after the initiation of chemotherapy. A plausible explanation is that MS developing at extramedullary sites requires more oncogenetic mutations.9 The high-intensity chemotherapy given to this patient may have accelerated the mutations that led to the development of MS as a second tumor.20–24 This patient had the abnormal chromosomal karyotype of 46, XY, −9, der (16) t (9; 16) (q13; q12), +mar. However, no evidence suggests that this chromosomal abnormality contributes to an increased susceptibility to oncogenic clonal evolution. Whether these chromosomal abnormalities increased the risk for the development of secondary tumors requires further investigation. Another hypothesis of the cause of MS was the cross-lineage transdifferentiation of B cell progenitors in BLBL to MS. In adults, transdifferentiations of BALL or other lymphoid progenitors into neoplasms of other lineage such as histiocytic sarcoma/leukemia have been reported in few cases.25–28 Transdifferentiations between neoplasms of tumor cells from other lineages have also been reported in sporadic adult cases.29,30 Most of these patients feature chromosomal and genetic abnormalities including the mutation of RAS genes. The presence of NRAS and KRAS genes and the expression of MPO may suggest the potential of transdifferentiation of B cells into myeloid blasts. However, transdifferentiation of BLBL to MS has not been reported in pediatric patients and needs further investigations. Conclusions In conclusion, we report a boy who developed MS after chemotherapy for BLBL after a period of approximately eight months. The exact mechanism of the development of MS in this patient remains uncertain. A diagnosis of BLBL was considered despite the expression of myeloid marker MPO. This patient exhibited certain cytogenetic abnormalities, but fusion genes were not presented. NGS evaluations revealed mutations of both NRAS and KRAS genes that may contribute to the genetic origin of MS. Therefore, understanding the mechanisms of the development of MS in this patient may help with the prediction of prognosis and the creation and advancement of therapeutic strategies for targeted treatments, and prevention of secondary malignancies. Acknowledgments We thank A/Prof Ronald Grant from the Hospital for Sick Children in Toronto, Canada for the long-time support to help us to provide better care for our pediatric oncology patients. Xiuli Yuan and Uet Yu are co-first authors for this study. Abbreviations 6-MP, 6-mercaptopurine; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; Ara-C, cytosine arabinoside; BLBL, B cell lymphoblastic lymphoma; CTX, cyclophosphamide; Dex, dexamethasone; DNR, daunoblastina; L-Peg-Asp, L-asparaginase; MPO, myeloperoxidase; MS, myeloid sarcoma; MTX, methotrexate; VCR, vincristine. Data Sharing Statement Not applicable. Ethical Approval and Informed Consent Ethical approval is not required for this Case report. Written informed consent was obtained from the patient’s parent. Consent for Publication Written informed consent was obtained from the patient’s parent for publication of this paper and any accompanying images. Author Contributions All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. Disclosure The authors have no competing interests to declare.	UNK UNK, CYCLIC	DrugDosageText	CC BY-NC	33469311	19,436,569	2021
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug ineffective'.	Pyogenic Liver Abscess and Endogenous Endophthalmitis Due to K64-ST1764 Hypervirulent Klebsiella pneumoniae: A Case Report. Klebsiella pneumoniae (K. pneumoniae, KP) are divided into two types: classic K. pneumoniae (cKP) and hypervirulent K. pneumoniae (hvKP). hvKP causes liver abscess and metastatic infection. Here, we report one case with pyogenic liver abscess (PLA) and endogenous endophthalmitis (EE) due to a relatively rarely reported serotype of K. pneumoniae in China. An 80-year old man presented with nausea, vomiting, and epigastric discomfort for 2 weeks. PLA was identified by CT scan and abdominal ultrasound. Urgent ophthalmologic consultation was performed. B-scan ocular ultrasound was done and he was diagnosed as EE. Antibiotic treatment, intravitreal injection of eyes and eye drops were given. Percutaneous needle aspiration, evisceration, and drainage of the right eye were performed. Cultures of the blood, the aspirated pus from the liver abscess, and the contents of the eyeball all yielded K. pneumoniae with a positive string test. The capsular serotype was K64. According to the existence of multiple virulence genes and the severe invasive clinical manifestation, this strain is regarded as a hvKp strain. Multilocus sequence typing (MLST) revealed the sequence type (ST) of this strain was K64-ST1764. Antimicrobial resistance genes, bla NDM-1 and bla KPC-2, were not detected in the genome. The patient lost his eyesight but his symptoms subsided. During 15 months follow-up, the result was satisfactory. Here, we report one case with PLA due to a relatively rarely reported serotype of K. pneumoniae in China. This K64 K. pneumoniae strain is confirmed as hvKp by multiple methods. It is noteworthy that the sequence type is K64-ST1764 instead of the commonest ST11. Moreover, this strain is not considered a K. pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) or a carbapenem-resistant K. pneumoniae (CRKP) as it is usually. Further follow-up and research are required to investigate this strain. Introduction PLA is a potentially life-threatening disease which is mainly caused by bacteria. Along with the increasing prevalence of hvKp, PLA due to K. pneumoniae has been steadily increasing. K. pneumoniae are divided into two types: cKP and hvKP.1 Ckp is an opportunistic pathogen which mainly infects immunodeficient patients or the immunocompromised population. HvKP can infect young healthy people without underlying diseases.2 Increased virulence of hvkp is related to its hypermucoviscous phenotype and stronger ability of iron acquisition.3,4 HvKP often causes primary liver abscess and metastatic infections like endophthalmitis, meningitis, and so on.5 HvKP infections are more common in the Asian Paciﬁc but now are occurring globally. Here we report one case who presented in China with PLA and EE due to a relatively rarely reported serotype of hvKP. Different from previous reports, the ST of this strain was K64-ST1764 instead of the commonest ST11. It is especially noteworthy that antimicrobial susceptibility testing showed that this strain was sensitive to all antibiotics, which means it is not a KPC-Kp or a CRKP. Case Presentation An 80-year old Chinese male patient presented to the emergency department with nausea, vomiting, and epigastric discomfort for 2 weeks without treatment. This patient had a history of Type II diabetes mellitus, hypertension, and coronary heart disease. There was no relevant family history or history of genetic disease. Laboratory investigations revealed a white cell count 14.42×109 cells/L (normal range=3.5–9.5×109 cells/L), 85% neutrophils (normal range=40–75%), aspartate transaminase (AST) 109 U/L (normal range<50 U/L), lactate dehydrogenase (LDH) 349 U/L (normal range=109–245 U/L), random blood glucose 19.96 mmol/L (normal range=3.8–6.2 mmol/L), blood urea 11.3 mmol/L (normal range=2.1–8.6 mmol/L), creatinine 141.7 µmol/L (normal range=35.2–97.5 μmol/L). Severe infection and ketoacidosis were suspected so emergency doctors gave him antibiotics (imipenem combined with ornidazole), rabeprazole, antiemetic drug, and fluid infusion, but his condition worsened. Fever appeared and the highest armpit temperature was 39°C. He was admitted to the Department of Gastroenterology for further treatment. Laboratory investigations revealed C-reactive protein 439.43 mg/L (normal range<10 mg/L), serum sodium (Na) 133.9 mmol/L (normal range=136–145 mmol/L), serum potassium (K) 3.71 mmol/L (normal range=3.5–5.3 mmol/L), total bilirubin 21 μmol/L (normal range=3.4–20.6 μmol/L), and alanine aminotransferase (ALT) 88.6 U/L (normal range<50 U/L). Physical examination revealed mild epigastric tenderness. A right lobe liver abscess was identified on CT scan (Figure 1). Abdominal ultrasound showed a right lobe liver abscess (mixed echoes) with the size of 3.5×2.7 cm in the right liver lobe (Figure 2).Figure 1 Abdominal CT scan showed a right lobe liver abscess (rounded, low density areas). Figure 2 Abdominal ultrasound showed a right lobe liver abscess (mixed echoes, with a size of 3.5cm×2.7 cm). The patient was treated by anti-infection treatment (biapenem and morinidazole by intravenous injection), liver protection (polyene phosphatidylcholine), decreasing blood sugar, controlling blood pressure, and fluid infusion. Ultrasonography guided puncture aspiration for hepatic abscess was proposed but he refused. Both his white cell count and C-reactive protein were dropped after treatment but he was still feverish. Bacteria culturing and antimicrobial susceptibility testing were performed by using the VITEK-II Compact automated microbiological system and confirmed by using the broth microdilution method. Blood cultures, from both aerobic and anaerobic bottles, showed K. pneumoniae which was sensitive to all antibiotics. The vision of this patient became blurred so urgent ophthalmologic consultation was performed. A B-scan ocular ultrasound was done and he was diagnosed as EE with bad prognosis. Intravitreal injection (vancomycin and cefoperazone) of eyes and eye drops were given. Finally, after obtaining the patient’s consent, ultrasonography guided puncture aspiration was performed, which resulted in lowering the temperature and improving the inﬂammatory marker levels. Liver aspirate culture was also positive for K. pneumoniae and sensitive to all antibiotics (Table 1). The patient’s eyes were swollen and empyema and the light perception of both eyes was basically lost. The ophthalmologist performed evisceration and drainage of the right eye, as well as repeated intravitreal injection of the left eye. Abdominal ultrasonography was performed 6 days after the first therapeutic puncture. The abscess cavity decreased conspicuously and the second percutaneous needle aspiration was given. After treatment, the patient’s eye swelling and the empyema was significantly improved and he became afebrile. He was given ceftriaxon 2 g by intravenous injection once a day for 4 weeks. Repeated blood cultures were negative and ultrosound reexamination showed the abscess cavity had disappeared. He was discharged and referred to the gastroenterology clinic for follow-up. During 15 months follow-up, the result was satisfactory.Table 1 Antimicrobial Susceptibility Testing Results for K. Pneumoniae Isolate, Using Reference Broth Microdilution According to Clinical and Laboratory Standards Institute (CLSI) Guidelines Antimicrobial Agent Minimum Inhibitory Concentration (µg/mL) Interpretation* Amikacin ≤2 S Ampicillin >32 S Aztreonam ≤1 S Ceftriaxone ≤1 S Ceftazidime ≤1 S Cefepime ≤1 S Ciprofloxacin ≤0.25 S Gentamicin ≤1 S Imipenem ≤1 S Levofloxacin ≤0.25 S Ertapenem ≤0.5 S Piperacillin-Tazobactam ≤4 S Tigecycline 0.5 S Notes: *Interpretative criteria were applied according to CLSI document M100 (Performance Standards for Antimicrobial Susceptibility Testing. 28th ed. 2018) except for tigecycline where criteria established by the US Food and Drug Administration were used. Abbreviations: R, resistant; S, susceptible. Isolates were retested by 16S rRNA gene sequences as previously reported6 and K. pneumoniae was identified and reconfirmed. String test was performed as previously reported.2 The colony was grown at 37°C overnight on a sheep blood agar plate and then stretched by a bacteriology inoculation loop. A positive result is defined as the formation of a string >5 mm in length. Cultures of the blood, the aspirated pus from the liver abscess, and the contents of the eyeball all yielded K. pneumoniae with a positive string test (Figure 3).Figure 3 All cultures yielded K. pneumoniae with a positive string test (>5mm in string length). Capsular serotype of each isolate was performed by polymerase chain reaction (PCR) as previously reported.7 PCR analysis indicated that the capsular serotype of the isolated K. pneumoniae was K64, which was validated by wzi sequencing as described.8 MLST was performed by PCR amplification as previously reported9 and this strain belongs to ST1764. Multiple biomarkers, including peg-344 (+), iroB (+), iucA (+), prmpA (+), prmpA2 (+), crmpA (-), ureA (+), uge (+), wabG (+), allS (+), mrkD (+), and fimH (+) were detected by PCR as described10 and multiple virulence genes were found in all the above isolates. Screening for carbapenemase genes was performed by PCR as described11 and blaNDM-1 and blaKPC-2 were not found in this K64 strain. We evaluated the virulence of this K. pneumoniae strain in a Galleria mellonella infection model.12 We infected the Galleria mellonella larvae with different concentrations (102~108 colony-forming units/mL) of K. pneumoniae and recorded the survival rate (Figure 4). This strain showed a strong concentration-dependent lethality to the larvae.Figure 4 Survival curves for Galleria mellonella larvae inoculated with different concentrations (102~108 colony-forming units/mL) of the K. pneumoniae strain. Discussion and Conclusions The ﬁrst clinical report about hvKp was in Taiwan in 1986.13 Although it is more common in the Asia-Pacific region, infections due to hvKp have gradually become global.14,15 Because hvKP was considered to have a hypermucoviscosity phenotype, the string test has been used to deﬁning hvKP.3 However, more and more studies have shown that hypermucoviscosity is not pathognomonic for hvKp since this phenotype also can be observed in cKp strains.16 Recently, several virulence genes, like iuc, peg-344, rmpA, and rmpA2, have been shown to most accurately predict hvKp strains.16 Since the 1980s, PLA due to K. pneumoniae has been steadily increasing. Especially in the Asia-Pacific region, hvKP has already become the predominant pathogen of PLA.17–19 Furthermore, these infections are occurring globally. A study in China has reported that among all PLA cases, K pneumonia was the most common pathogen and a higher incidence of diabetes mellitus was shown in patients with K. pneumoniae-induced PLA.20 Another study has reported that most of the liver abscesses were caused by K. pneumoniae and mostly occurred in patients with diabetes mellitus.21 The major clinical features of KP-PLA are fever, nausea, vomiting, abdominal discomfort, and laboratory inspection shows elevated white blood cell count and liver dysfunction. In this patient, all the above features can be found. There are mainly three treatment methods: appropriate early antibiotic treatment, ultrasonography guided puncture aspiration or draining treatment, and open surgical drainage or surgical resection of the liver abscesses. We initially treated the patient with carbapenems and Morinidazole. After the condition stabilized, the antibiotics were changed to ceftriaxone for another 4 weeks. Some studies have suggested that the majority of PLA cases require drainage.22 A report showed that antimicrobial therapy alone was effective for small abscesses, while percutaneous needle aspiration or draining should be chosen for larger abscesses.23 A study reported that liver abscesses due to K. pneumoniae that were >5 cm were likely to have a delayed response to therapy.24 In the present study, the liver abscess was <5 cm (35×27 mm) and this hvKP strain was sensitive to all antibiotics. Even so, early antimicrobial therapy alone was ineffective. Once aspiration was performed, clinical improvement was observed. This suggested that, even for small abscesses, puncture or drainage should be performed as early as possible when the effect of drug therapy is unsatisfactory. In recent years, the incidence of EE has gradually increased. Gram-positive bacteria and fungal organisms account for the majority of the cases in the developed world, whereas gram-negative organisms like K. pneumoniae are more common in the Asia-Paciﬁc region.25 Now it has become a frequent complication: approximately 5% of individuals with hvKp bacteremia ultimately develop EE.26 EE is a common metastatic complication of K. pneumoniae liver abscess.27 Diabetes mellitus was considered to be a significant risk factor. The prognosis of EE is generally poor even with prompt diagnosis and aggressive treatment given. The ﬁnal visual outcome was only light perception or worse in 89% of patients with EE associated with K. pneumoniae-induced PLA.27 Diabetes mellitus and poor initial visual acuity were associated with poor visual outcome.28 Reports showed that the main prognostic factor in Klebsiella EE is the presence of hypopyon which can be found in our patient.29 This patient lost his eyesight. It has long been recognized that almost all patients with severe infection with liver abscess and extrahepatic infections are infected exclusively with K. pneumoniae serotypes K1 or K2.2 However, it is clear now that non-K1/K2 hvKp strains can express virulence genes and are capable of causing liver abscess and metastatic spread.16,30 The serotype of the isolated K. pneumoniae strain in our patient was K64, a relatively rarely reported serotype. This serotype has always been considered a cKP. But it is recently recognized that K64 is predicted to be observed for hvKp strains.31,32 Recent research finds that some cKp strains can acquire the hvKp virulence plasmid and evolve into hvKp strains.12,32 Our K64 K. pneumoniae strain showed strong virulence in the Galleria mellonella infection model. According to positive string test, the existence of multiple virulence genes, the strong lethality to the larvae and the severe invasive clinical manifestation of its infection, we can regard this K64 strain as a hvKp strain. Compared to the classification based on string test alone, our method is more reasonable. In particular, ST was determined by MLST and the result was K64-ST1764 instead of the commonest ST11, which was considered to be predominant in Extended-Spectrum β-lactamases (ESBL)-producing strains and was prevalent in CRKP strains.33,34 However, previous reports showed that ST11 was predominant in K64 capsular type, hence K64 was considered as a prevalent type of CRKP.35–38 Furthermore, blaNDM-1 and blaKPC-2, two important carbapenemase genes, were not found in this K64 strain and antimicrobial susceptibility testing showed that it was sensitive to all antibiotics. Contrary to previous studies,31,32,36,39 these findings indicated that this K64 strain is not a KPC-Kp or a CRKP strain. In conclusion, here we report one patient with PLA and EE due to a relatively rarely reported serotype of K. pneumoniae: K64. As the existence of multiple virulence genes, the strong lethality to the larvae and the severe invasive clinical manifestation and positive string test, this K64 K. pneumoniae can be regarded as a hvKp strain. Different from previous reports, the ST of this strain was K64-ST1764 instead of the commonest ST11. It is not a KPC-Kp or a CRKP, either. Further follow-up and research are required to investigate this strain. Acknowledgments The key discipline of Wuxi No.ZDXK002 Workstation of academician Fan Daiming No.CYR1705 Abbreviations CRKP, carbapenem-resistant K. pneumoniae; cKP, classic K. pneumoniae; ESBL, extended-spectrum β-lactamases; hvKP, hypervirulent K. pneumoniae; KPC-Kp, K. pneumoniae carbapenemase (KPC)-producing ; MLST, multilocus sequence typing; PLA, pyogenic liver abscess; ST, sequence type. Data Sharing Statement The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics Approval This case report was approved by the ethics committee of The Afﬁliated Wuxi Second People’s Hospital of Nanjing Medical University. Consent for Publication Written informed consent for publication of his clinical details and clinical images was obtained from the patient. Author Contributions Bo Zhao and Renjing Hu have contributed equally to this work and they are co-first authors. Conceived the study: Xiaoyun Wang, Bo Zhao; Acquired data, analyzed data: Bo Zhao, Renjing Hu; Performed the microbiological analysis: Renjing Hu; Wrote the manuscript: Bo Zhao; Contributed reagents/materials/analysis tools: Lei Gong, Xiaoyun Wang; and Revised the manuscript: Xiaoyun Wang, Yingwei Zhu, Gaojue Wu. All authors made substantial contributions to the conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. Disclosure The authors declare that they have no conflicts of interest to this work.	BIAPENEM, CEFOPERAZONE, PHOSPHOLIPID, VANCOMYCIN	DrugsGivenReaction	CC BY-NC	33469321	19,979,976	2021
What was the administration route of drug 'BIAPENEM'?	Pyogenic Liver Abscess and Endogenous Endophthalmitis Due to K64-ST1764 Hypervirulent Klebsiella pneumoniae: A Case Report. Klebsiella pneumoniae (K. pneumoniae, KP) are divided into two types: classic K. pneumoniae (cKP) and hypervirulent K. pneumoniae (hvKP). hvKP causes liver abscess and metastatic infection. Here, we report one case with pyogenic liver abscess (PLA) and endogenous endophthalmitis (EE) due to a relatively rarely reported serotype of K. pneumoniae in China. An 80-year old man presented with nausea, vomiting, and epigastric discomfort for 2 weeks. PLA was identified by CT scan and abdominal ultrasound. Urgent ophthalmologic consultation was performed. B-scan ocular ultrasound was done and he was diagnosed as EE. Antibiotic treatment, intravitreal injection of eyes and eye drops were given. Percutaneous needle aspiration, evisceration, and drainage of the right eye were performed. Cultures of the blood, the aspirated pus from the liver abscess, and the contents of the eyeball all yielded K. pneumoniae with a positive string test. The capsular serotype was K64. According to the existence of multiple virulence genes and the severe invasive clinical manifestation, this strain is regarded as a hvKp strain. Multilocus sequence typing (MLST) revealed the sequence type (ST) of this strain was K64-ST1764. Antimicrobial resistance genes, bla NDM-1 and bla KPC-2, were not detected in the genome. The patient lost his eyesight but his symptoms subsided. During 15 months follow-up, the result was satisfactory. Here, we report one case with PLA due to a relatively rarely reported serotype of K. pneumoniae in China. This K64 K. pneumoniae strain is confirmed as hvKp by multiple methods. It is noteworthy that the sequence type is K64-ST1764 instead of the commonest ST11. Moreover, this strain is not considered a K. pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) or a carbapenem-resistant K. pneumoniae (CRKP) as it is usually. Further follow-up and research are required to investigate this strain. Introduction PLA is a potentially life-threatening disease which is mainly caused by bacteria. Along with the increasing prevalence of hvKp, PLA due to K. pneumoniae has been steadily increasing. K. pneumoniae are divided into two types: cKP and hvKP.1 Ckp is an opportunistic pathogen which mainly infects immunodeficient patients or the immunocompromised population. HvKP can infect young healthy people without underlying diseases.2 Increased virulence of hvkp is related to its hypermucoviscous phenotype and stronger ability of iron acquisition.3,4 HvKP often causes primary liver abscess and metastatic infections like endophthalmitis, meningitis, and so on.5 HvKP infections are more common in the Asian Paciﬁc but now are occurring globally. Here we report one case who presented in China with PLA and EE due to a relatively rarely reported serotype of hvKP. Different from previous reports, the ST of this strain was K64-ST1764 instead of the commonest ST11. It is especially noteworthy that antimicrobial susceptibility testing showed that this strain was sensitive to all antibiotics, which means it is not a KPC-Kp or a CRKP. Case Presentation An 80-year old Chinese male patient presented to the emergency department with nausea, vomiting, and epigastric discomfort for 2 weeks without treatment. This patient had a history of Type II diabetes mellitus, hypertension, and coronary heart disease. There was no relevant family history or history of genetic disease. Laboratory investigations revealed a white cell count 14.42×109 cells/L (normal range=3.5–9.5×109 cells/L), 85% neutrophils (normal range=40–75%), aspartate transaminase (AST) 109 U/L (normal range<50 U/L), lactate dehydrogenase (LDH) 349 U/L (normal range=109–245 U/L), random blood glucose 19.96 mmol/L (normal range=3.8–6.2 mmol/L), blood urea 11.3 mmol/L (normal range=2.1–8.6 mmol/L), creatinine 141.7 µmol/L (normal range=35.2–97.5 μmol/L). Severe infection and ketoacidosis were suspected so emergency doctors gave him antibiotics (imipenem combined with ornidazole), rabeprazole, antiemetic drug, and fluid infusion, but his condition worsened. Fever appeared and the highest armpit temperature was 39°C. He was admitted to the Department of Gastroenterology for further treatment. Laboratory investigations revealed C-reactive protein 439.43 mg/L (normal range<10 mg/L), serum sodium (Na) 133.9 mmol/L (normal range=136–145 mmol/L), serum potassium (K) 3.71 mmol/L (normal range=3.5–5.3 mmol/L), total bilirubin 21 μmol/L (normal range=3.4–20.6 μmol/L), and alanine aminotransferase (ALT) 88.6 U/L (normal range<50 U/L). Physical examination revealed mild epigastric tenderness. A right lobe liver abscess was identified on CT scan (Figure 1). Abdominal ultrasound showed a right lobe liver abscess (mixed echoes) with the size of 3.5×2.7 cm in the right liver lobe (Figure 2).Figure 1 Abdominal CT scan showed a right lobe liver abscess (rounded, low density areas). Figure 2 Abdominal ultrasound showed a right lobe liver abscess (mixed echoes, with a size of 3.5cm×2.7 cm). The patient was treated by anti-infection treatment (biapenem and morinidazole by intravenous injection), liver protection (polyene phosphatidylcholine), decreasing blood sugar, controlling blood pressure, and fluid infusion. Ultrasonography guided puncture aspiration for hepatic abscess was proposed but he refused. Both his white cell count and C-reactive protein were dropped after treatment but he was still feverish. Bacteria culturing and antimicrobial susceptibility testing were performed by using the VITEK-II Compact automated microbiological system and confirmed by using the broth microdilution method. Blood cultures, from both aerobic and anaerobic bottles, showed K. pneumoniae which was sensitive to all antibiotics. The vision of this patient became blurred so urgent ophthalmologic consultation was performed. A B-scan ocular ultrasound was done and he was diagnosed as EE with bad prognosis. Intravitreal injection (vancomycin and cefoperazone) of eyes and eye drops were given. Finally, after obtaining the patient’s consent, ultrasonography guided puncture aspiration was performed, which resulted in lowering the temperature and improving the inﬂammatory marker levels. Liver aspirate culture was also positive for K. pneumoniae and sensitive to all antibiotics (Table 1). The patient’s eyes were swollen and empyema and the light perception of both eyes was basically lost. The ophthalmologist performed evisceration and drainage of the right eye, as well as repeated intravitreal injection of the left eye. Abdominal ultrasonography was performed 6 days after the first therapeutic puncture. The abscess cavity decreased conspicuously and the second percutaneous needle aspiration was given. After treatment, the patient’s eye swelling and the empyema was significantly improved and he became afebrile. He was given ceftriaxon 2 g by intravenous injection once a day for 4 weeks. Repeated blood cultures were negative and ultrosound reexamination showed the abscess cavity had disappeared. He was discharged and referred to the gastroenterology clinic for follow-up. During 15 months follow-up, the result was satisfactory.Table 1 Antimicrobial Susceptibility Testing Results for K. Pneumoniae Isolate, Using Reference Broth Microdilution According to Clinical and Laboratory Standards Institute (CLSI) Guidelines Antimicrobial Agent Minimum Inhibitory Concentration (µg/mL) Interpretation* Amikacin ≤2 S Ampicillin >32 S Aztreonam ≤1 S Ceftriaxone ≤1 S Ceftazidime ≤1 S Cefepime ≤1 S Ciprofloxacin ≤0.25 S Gentamicin ≤1 S Imipenem ≤1 S Levofloxacin ≤0.25 S Ertapenem ≤0.5 S Piperacillin-Tazobactam ≤4 S Tigecycline 0.5 S Notes: *Interpretative criteria were applied according to CLSI document M100 (Performance Standards for Antimicrobial Susceptibility Testing. 28th ed. 2018) except for tigecycline where criteria established by the US Food and Drug Administration were used. Abbreviations: R, resistant; S, susceptible. Isolates were retested by 16S rRNA gene sequences as previously reported6 and K. pneumoniae was identified and reconfirmed. String test was performed as previously reported.2 The colony was grown at 37°C overnight on a sheep blood agar plate and then stretched by a bacteriology inoculation loop. A positive result is defined as the formation of a string >5 mm in length. Cultures of the blood, the aspirated pus from the liver abscess, and the contents of the eyeball all yielded K. pneumoniae with a positive string test (Figure 3).Figure 3 All cultures yielded K. pneumoniae with a positive string test (>5mm in string length). Capsular serotype of each isolate was performed by polymerase chain reaction (PCR) as previously reported.7 PCR analysis indicated that the capsular serotype of the isolated K. pneumoniae was K64, which was validated by wzi sequencing as described.8 MLST was performed by PCR amplification as previously reported9 and this strain belongs to ST1764. Multiple biomarkers, including peg-344 (+), iroB (+), iucA (+), prmpA (+), prmpA2 (+), crmpA (-), ureA (+), uge (+), wabG (+), allS (+), mrkD (+), and fimH (+) were detected by PCR as described10 and multiple virulence genes were found in all the above isolates. Screening for carbapenemase genes was performed by PCR as described11 and blaNDM-1 and blaKPC-2 were not found in this K64 strain. We evaluated the virulence of this K. pneumoniae strain in a Galleria mellonella infection model.12 We infected the Galleria mellonella larvae with different concentrations (102~108 colony-forming units/mL) of K. pneumoniae and recorded the survival rate (Figure 4). This strain showed a strong concentration-dependent lethality to the larvae.Figure 4 Survival curves for Galleria mellonella larvae inoculated with different concentrations (102~108 colony-forming units/mL) of the K. pneumoniae strain. Discussion and Conclusions The ﬁrst clinical report about hvKp was in Taiwan in 1986.13 Although it is more common in the Asia-Pacific region, infections due to hvKp have gradually become global.14,15 Because hvKP was considered to have a hypermucoviscosity phenotype, the string test has been used to deﬁning hvKP.3 However, more and more studies have shown that hypermucoviscosity is not pathognomonic for hvKp since this phenotype also can be observed in cKp strains.16 Recently, several virulence genes, like iuc, peg-344, rmpA, and rmpA2, have been shown to most accurately predict hvKp strains.16 Since the 1980s, PLA due to K. pneumoniae has been steadily increasing. Especially in the Asia-Pacific region, hvKP has already become the predominant pathogen of PLA.17–19 Furthermore, these infections are occurring globally. A study in China has reported that among all PLA cases, K pneumonia was the most common pathogen and a higher incidence of diabetes mellitus was shown in patients with K. pneumoniae-induced PLA.20 Another study has reported that most of the liver abscesses were caused by K. pneumoniae and mostly occurred in patients with diabetes mellitus.21 The major clinical features of KP-PLA are fever, nausea, vomiting, abdominal discomfort, and laboratory inspection shows elevated white blood cell count and liver dysfunction. In this patient, all the above features can be found. There are mainly three treatment methods: appropriate early antibiotic treatment, ultrasonography guided puncture aspiration or draining treatment, and open surgical drainage or surgical resection of the liver abscesses. We initially treated the patient with carbapenems and Morinidazole. After the condition stabilized, the antibiotics were changed to ceftriaxone for another 4 weeks. Some studies have suggested that the majority of PLA cases require drainage.22 A report showed that antimicrobial therapy alone was effective for small abscesses, while percutaneous needle aspiration or draining should be chosen for larger abscesses.23 A study reported that liver abscesses due to K. pneumoniae that were >5 cm were likely to have a delayed response to therapy.24 In the present study, the liver abscess was <5 cm (35×27 mm) and this hvKP strain was sensitive to all antibiotics. Even so, early antimicrobial therapy alone was ineffective. Once aspiration was performed, clinical improvement was observed. This suggested that, even for small abscesses, puncture or drainage should be performed as early as possible when the effect of drug therapy is unsatisfactory. In recent years, the incidence of EE has gradually increased. Gram-positive bacteria and fungal organisms account for the majority of the cases in the developed world, whereas gram-negative organisms like K. pneumoniae are more common in the Asia-Paciﬁc region.25 Now it has become a frequent complication: approximately 5% of individuals with hvKp bacteremia ultimately develop EE.26 EE is a common metastatic complication of K. pneumoniae liver abscess.27 Diabetes mellitus was considered to be a significant risk factor. The prognosis of EE is generally poor even with prompt diagnosis and aggressive treatment given. The ﬁnal visual outcome was only light perception or worse in 89% of patients with EE associated with K. pneumoniae-induced PLA.27 Diabetes mellitus and poor initial visual acuity were associated with poor visual outcome.28 Reports showed that the main prognostic factor in Klebsiella EE is the presence of hypopyon which can be found in our patient.29 This patient lost his eyesight. It has long been recognized that almost all patients with severe infection with liver abscess and extrahepatic infections are infected exclusively with K. pneumoniae serotypes K1 or K2.2 However, it is clear now that non-K1/K2 hvKp strains can express virulence genes and are capable of causing liver abscess and metastatic spread.16,30 The serotype of the isolated K. pneumoniae strain in our patient was K64, a relatively rarely reported serotype. This serotype has always been considered a cKP. But it is recently recognized that K64 is predicted to be observed for hvKp strains.31,32 Recent research finds that some cKp strains can acquire the hvKp virulence plasmid and evolve into hvKp strains.12,32 Our K64 K. pneumoniae strain showed strong virulence in the Galleria mellonella infection model. According to positive string test, the existence of multiple virulence genes, the strong lethality to the larvae and the severe invasive clinical manifestation of its infection, we can regard this K64 strain as a hvKp strain. Compared to the classification based on string test alone, our method is more reasonable. In particular, ST was determined by MLST and the result was K64-ST1764 instead of the commonest ST11, which was considered to be predominant in Extended-Spectrum β-lactamases (ESBL)-producing strains and was prevalent in CRKP strains.33,34 However, previous reports showed that ST11 was predominant in K64 capsular type, hence K64 was considered as a prevalent type of CRKP.35–38 Furthermore, blaNDM-1 and blaKPC-2, two important carbapenemase genes, were not found in this K64 strain and antimicrobial susceptibility testing showed that it was sensitive to all antibiotics. Contrary to previous studies,31,32,36,39 these findings indicated that this K64 strain is not a KPC-Kp or a CRKP strain. In conclusion, here we report one patient with PLA and EE due to a relatively rarely reported serotype of K. pneumoniae: K64. As the existence of multiple virulence genes, the strong lethality to the larvae and the severe invasive clinical manifestation and positive string test, this K64 K. pneumoniae can be regarded as a hvKp strain. Different from previous reports, the ST of this strain was K64-ST1764 instead of the commonest ST11. It is not a KPC-Kp or a CRKP, either. Further follow-up and research are required to investigate this strain. Acknowledgments The key discipline of Wuxi No.ZDXK002 Workstation of academician Fan Daiming No.CYR1705 Abbreviations CRKP, carbapenem-resistant K. pneumoniae; cKP, classic K. pneumoniae; ESBL, extended-spectrum β-lactamases; hvKP, hypervirulent K. pneumoniae; KPC-Kp, K. pneumoniae carbapenemase (KPC)-producing ; MLST, multilocus sequence typing; PLA, pyogenic liver abscess; ST, sequence type. Data Sharing Statement The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics Approval This case report was approved by the ethics committee of The Afﬁliated Wuxi Second People’s Hospital of Nanjing Medical University. Consent for Publication Written informed consent for publication of his clinical details and clinical images was obtained from the patient. Author Contributions Bo Zhao and Renjing Hu have contributed equally to this work and they are co-first authors. Conceived the study: Xiaoyun Wang, Bo Zhao; Acquired data, analyzed data: Bo Zhao, Renjing Hu; Performed the microbiological analysis: Renjing Hu; Wrote the manuscript: Bo Zhao; Contributed reagents/materials/analysis tools: Lei Gong, Xiaoyun Wang; and Revised the manuscript: Xiaoyun Wang, Yingwei Zhu, Gaojue Wu. All authors made substantial contributions to the conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. Disclosure The authors declare that they have no conflicts of interest to this work.	Intravenous (not otherwise specified)	DrugAdministrationRoute	CC BY-NC	33469321	19,979,976	2021
What was the dosage of drug 'VANCOMYCIN'?	Pyogenic Liver Abscess and Endogenous Endophthalmitis Due to K64-ST1764 Hypervirulent Klebsiella pneumoniae: A Case Report. Klebsiella pneumoniae (K. pneumoniae, KP) are divided into two types: classic K. pneumoniae (cKP) and hypervirulent K. pneumoniae (hvKP). hvKP causes liver abscess and metastatic infection. Here, we report one case with pyogenic liver abscess (PLA) and endogenous endophthalmitis (EE) due to a relatively rarely reported serotype of K. pneumoniae in China. An 80-year old man presented with nausea, vomiting, and epigastric discomfort for 2 weeks. PLA was identified by CT scan and abdominal ultrasound. Urgent ophthalmologic consultation was performed. B-scan ocular ultrasound was done and he was diagnosed as EE. Antibiotic treatment, intravitreal injection of eyes and eye drops were given. Percutaneous needle aspiration, evisceration, and drainage of the right eye were performed. Cultures of the blood, the aspirated pus from the liver abscess, and the contents of the eyeball all yielded K. pneumoniae with a positive string test. The capsular serotype was K64. According to the existence of multiple virulence genes and the severe invasive clinical manifestation, this strain is regarded as a hvKp strain. Multilocus sequence typing (MLST) revealed the sequence type (ST) of this strain was K64-ST1764. Antimicrobial resistance genes, bla NDM-1 and bla KPC-2, were not detected in the genome. The patient lost his eyesight but his symptoms subsided. During 15 months follow-up, the result was satisfactory. Here, we report one case with PLA due to a relatively rarely reported serotype of K. pneumoniae in China. This K64 K. pneumoniae strain is confirmed as hvKp by multiple methods. It is noteworthy that the sequence type is K64-ST1764 instead of the commonest ST11. Moreover, this strain is not considered a K. pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) or a carbapenem-resistant K. pneumoniae (CRKP) as it is usually. Further follow-up and research are required to investigate this strain. Introduction PLA is a potentially life-threatening disease which is mainly caused by bacteria. Along with the increasing prevalence of hvKp, PLA due to K. pneumoniae has been steadily increasing. K. pneumoniae are divided into two types: cKP and hvKP.1 Ckp is an opportunistic pathogen which mainly infects immunodeficient patients or the immunocompromised population. HvKP can infect young healthy people without underlying diseases.2 Increased virulence of hvkp is related to its hypermucoviscous phenotype and stronger ability of iron acquisition.3,4 HvKP often causes primary liver abscess and metastatic infections like endophthalmitis, meningitis, and so on.5 HvKP infections are more common in the Asian Paciﬁc but now are occurring globally. Here we report one case who presented in China with PLA and EE due to a relatively rarely reported serotype of hvKP. Different from previous reports, the ST of this strain was K64-ST1764 instead of the commonest ST11. It is especially noteworthy that antimicrobial susceptibility testing showed that this strain was sensitive to all antibiotics, which means it is not a KPC-Kp or a CRKP. Case Presentation An 80-year old Chinese male patient presented to the emergency department with nausea, vomiting, and epigastric discomfort for 2 weeks without treatment. This patient had a history of Type II diabetes mellitus, hypertension, and coronary heart disease. There was no relevant family history or history of genetic disease. Laboratory investigations revealed a white cell count 14.42×109 cells/L (normal range=3.5–9.5×109 cells/L), 85% neutrophils (normal range=40–75%), aspartate transaminase (AST) 109 U/L (normal range<50 U/L), lactate dehydrogenase (LDH) 349 U/L (normal range=109–245 U/L), random blood glucose 19.96 mmol/L (normal range=3.8–6.2 mmol/L), blood urea 11.3 mmol/L (normal range=2.1–8.6 mmol/L), creatinine 141.7 µmol/L (normal range=35.2–97.5 μmol/L). Severe infection and ketoacidosis were suspected so emergency doctors gave him antibiotics (imipenem combined with ornidazole), rabeprazole, antiemetic drug, and fluid infusion, but his condition worsened. Fever appeared and the highest armpit temperature was 39°C. He was admitted to the Department of Gastroenterology for further treatment. Laboratory investigations revealed C-reactive protein 439.43 mg/L (normal range<10 mg/L), serum sodium (Na) 133.9 mmol/L (normal range=136–145 mmol/L), serum potassium (K) 3.71 mmol/L (normal range=3.5–5.3 mmol/L), total bilirubin 21 μmol/L (normal range=3.4–20.6 μmol/L), and alanine aminotransferase (ALT) 88.6 U/L (normal range<50 U/L). Physical examination revealed mild epigastric tenderness. A right lobe liver abscess was identified on CT scan (Figure 1). Abdominal ultrasound showed a right lobe liver abscess (mixed echoes) with the size of 3.5×2.7 cm in the right liver lobe (Figure 2).Figure 1 Abdominal CT scan showed a right lobe liver abscess (rounded, low density areas). Figure 2 Abdominal ultrasound showed a right lobe liver abscess (mixed echoes, with a size of 3.5cm×2.7 cm). The patient was treated by anti-infection treatment (biapenem and morinidazole by intravenous injection), liver protection (polyene phosphatidylcholine), decreasing blood sugar, controlling blood pressure, and fluid infusion. Ultrasonography guided puncture aspiration for hepatic abscess was proposed but he refused. Both his white cell count and C-reactive protein were dropped after treatment but he was still feverish. Bacteria culturing and antimicrobial susceptibility testing were performed by using the VITEK-II Compact automated microbiological system and confirmed by using the broth microdilution method. Blood cultures, from both aerobic and anaerobic bottles, showed K. pneumoniae which was sensitive to all antibiotics. The vision of this patient became blurred so urgent ophthalmologic consultation was performed. A B-scan ocular ultrasound was done and he was diagnosed as EE with bad prognosis. Intravitreal injection (vancomycin and cefoperazone) of eyes and eye drops were given. Finally, after obtaining the patient’s consent, ultrasonography guided puncture aspiration was performed, which resulted in lowering the temperature and improving the inﬂammatory marker levels. Liver aspirate culture was also positive for K. pneumoniae and sensitive to all antibiotics (Table 1). The patient’s eyes were swollen and empyema and the light perception of both eyes was basically lost. The ophthalmologist performed evisceration and drainage of the right eye, as well as repeated intravitreal injection of the left eye. Abdominal ultrasonography was performed 6 days after the first therapeutic puncture. The abscess cavity decreased conspicuously and the second percutaneous needle aspiration was given. After treatment, the patient’s eye swelling and the empyema was significantly improved and he became afebrile. He was given ceftriaxon 2 g by intravenous injection once a day for 4 weeks. Repeated blood cultures were negative and ultrosound reexamination showed the abscess cavity had disappeared. He was discharged and referred to the gastroenterology clinic for follow-up. During 15 months follow-up, the result was satisfactory.Table 1 Antimicrobial Susceptibility Testing Results for K. Pneumoniae Isolate, Using Reference Broth Microdilution According to Clinical and Laboratory Standards Institute (CLSI) Guidelines Antimicrobial Agent Minimum Inhibitory Concentration (µg/mL) Interpretation* Amikacin ≤2 S Ampicillin >32 S Aztreonam ≤1 S Ceftriaxone ≤1 S Ceftazidime ≤1 S Cefepime ≤1 S Ciprofloxacin ≤0.25 S Gentamicin ≤1 S Imipenem ≤1 S Levofloxacin ≤0.25 S Ertapenem ≤0.5 S Piperacillin-Tazobactam ≤4 S Tigecycline 0.5 S Notes: *Interpretative criteria were applied according to CLSI document M100 (Performance Standards for Antimicrobial Susceptibility Testing. 28th ed. 2018) except for tigecycline where criteria established by the US Food and Drug Administration were used. Abbreviations: R, resistant; S, susceptible. Isolates were retested by 16S rRNA gene sequences as previously reported6 and K. pneumoniae was identified and reconfirmed. String test was performed as previously reported.2 The colony was grown at 37°C overnight on a sheep blood agar plate and then stretched by a bacteriology inoculation loop. A positive result is defined as the formation of a string >5 mm in length. Cultures of the blood, the aspirated pus from the liver abscess, and the contents of the eyeball all yielded K. pneumoniae with a positive string test (Figure 3).Figure 3 All cultures yielded K. pneumoniae with a positive string test (>5mm in string length). Capsular serotype of each isolate was performed by polymerase chain reaction (PCR) as previously reported.7 PCR analysis indicated that the capsular serotype of the isolated K. pneumoniae was K64, which was validated by wzi sequencing as described.8 MLST was performed by PCR amplification as previously reported9 and this strain belongs to ST1764. Multiple biomarkers, including peg-344 (+), iroB (+), iucA (+), prmpA (+), prmpA2 (+), crmpA (-), ureA (+), uge (+), wabG (+), allS (+), mrkD (+), and fimH (+) were detected by PCR as described10 and multiple virulence genes were found in all the above isolates. Screening for carbapenemase genes was performed by PCR as described11 and blaNDM-1 and blaKPC-2 were not found in this K64 strain. We evaluated the virulence of this K. pneumoniae strain in a Galleria mellonella infection model.12 We infected the Galleria mellonella larvae with different concentrations (102~108 colony-forming units/mL) of K. pneumoniae and recorded the survival rate (Figure 4). This strain showed a strong concentration-dependent lethality to the larvae.Figure 4 Survival curves for Galleria mellonella larvae inoculated with different concentrations (102~108 colony-forming units/mL) of the K. pneumoniae strain. Discussion and Conclusions The ﬁrst clinical report about hvKp was in Taiwan in 1986.13 Although it is more common in the Asia-Pacific region, infections due to hvKp have gradually become global.14,15 Because hvKP was considered to have a hypermucoviscosity phenotype, the string test has been used to deﬁning hvKP.3 However, more and more studies have shown that hypermucoviscosity is not pathognomonic for hvKp since this phenotype also can be observed in cKp strains.16 Recently, several virulence genes, like iuc, peg-344, rmpA, and rmpA2, have been shown to most accurately predict hvKp strains.16 Since the 1980s, PLA due to K. pneumoniae has been steadily increasing. Especially in the Asia-Pacific region, hvKP has already become the predominant pathogen of PLA.17–19 Furthermore, these infections are occurring globally. A study in China has reported that among all PLA cases, K pneumonia was the most common pathogen and a higher incidence of diabetes mellitus was shown in patients with K. pneumoniae-induced PLA.20 Another study has reported that most of the liver abscesses were caused by K. pneumoniae and mostly occurred in patients with diabetes mellitus.21 The major clinical features of KP-PLA are fever, nausea, vomiting, abdominal discomfort, and laboratory inspection shows elevated white blood cell count and liver dysfunction. In this patient, all the above features can be found. There are mainly three treatment methods: appropriate early antibiotic treatment, ultrasonography guided puncture aspiration or draining treatment, and open surgical drainage or surgical resection of the liver abscesses. We initially treated the patient with carbapenems and Morinidazole. After the condition stabilized, the antibiotics were changed to ceftriaxone for another 4 weeks. Some studies have suggested that the majority of PLA cases require drainage.22 A report showed that antimicrobial therapy alone was effective for small abscesses, while percutaneous needle aspiration or draining should be chosen for larger abscesses.23 A study reported that liver abscesses due to K. pneumoniae that were >5 cm were likely to have a delayed response to therapy.24 In the present study, the liver abscess was <5 cm (35×27 mm) and this hvKP strain was sensitive to all antibiotics. Even so, early antimicrobial therapy alone was ineffective. Once aspiration was performed, clinical improvement was observed. This suggested that, even for small abscesses, puncture or drainage should be performed as early as possible when the effect of drug therapy is unsatisfactory. In recent years, the incidence of EE has gradually increased. Gram-positive bacteria and fungal organisms account for the majority of the cases in the developed world, whereas gram-negative organisms like K. pneumoniae are more common in the Asia-Paciﬁc region.25 Now it has become a frequent complication: approximately 5% of individuals with hvKp bacteremia ultimately develop EE.26 EE is a common metastatic complication of K. pneumoniae liver abscess.27 Diabetes mellitus was considered to be a significant risk factor. The prognosis of EE is generally poor even with prompt diagnosis and aggressive treatment given. The ﬁnal visual outcome was only light perception or worse in 89% of patients with EE associated with K. pneumoniae-induced PLA.27 Diabetes mellitus and poor initial visual acuity were associated with poor visual outcome.28 Reports showed that the main prognostic factor in Klebsiella EE is the presence of hypopyon which can be found in our patient.29 This patient lost his eyesight. It has long been recognized that almost all patients with severe infection with liver abscess and extrahepatic infections are infected exclusively with K. pneumoniae serotypes K1 or K2.2 However, it is clear now that non-K1/K2 hvKp strains can express virulence genes and are capable of causing liver abscess and metastatic spread.16,30 The serotype of the isolated K. pneumoniae strain in our patient was K64, a relatively rarely reported serotype. This serotype has always been considered a cKP. But it is recently recognized that K64 is predicted to be observed for hvKp strains.31,32 Recent research finds that some cKp strains can acquire the hvKp virulence plasmid and evolve into hvKp strains.12,32 Our K64 K. pneumoniae strain showed strong virulence in the Galleria mellonella infection model. According to positive string test, the existence of multiple virulence genes, the strong lethality to the larvae and the severe invasive clinical manifestation of its infection, we can regard this K64 strain as a hvKp strain. Compared to the classification based on string test alone, our method is more reasonable. In particular, ST was determined by MLST and the result was K64-ST1764 instead of the commonest ST11, which was considered to be predominant in Extended-Spectrum β-lactamases (ESBL)-producing strains and was prevalent in CRKP strains.33,34 However, previous reports showed that ST11 was predominant in K64 capsular type, hence K64 was considered as a prevalent type of CRKP.35–38 Furthermore, blaNDM-1 and blaKPC-2, two important carbapenemase genes, were not found in this K64 strain and antimicrobial susceptibility testing showed that it was sensitive to all antibiotics. Contrary to previous studies,31,32,36,39 these findings indicated that this K64 strain is not a KPC-Kp or a CRKP strain. In conclusion, here we report one patient with PLA and EE due to a relatively rarely reported serotype of K. pneumoniae: K64. As the existence of multiple virulence genes, the strong lethality to the larvae and the severe invasive clinical manifestation and positive string test, this K64 K. pneumoniae can be regarded as a hvKp strain. Different from previous reports, the ST of this strain was K64-ST1764 instead of the commonest ST11. It is not a KPC-Kp or a CRKP, either. Further follow-up and research are required to investigate this strain. Acknowledgments The key discipline of Wuxi No.ZDXK002 Workstation of academician Fan Daiming No.CYR1705 Abbreviations CRKP, carbapenem-resistant K. pneumoniae; cKP, classic K. pneumoniae; ESBL, extended-spectrum β-lactamases; hvKP, hypervirulent K. pneumoniae; KPC-Kp, K. pneumoniae carbapenemase (KPC)-producing ; MLST, multilocus sequence typing; PLA, pyogenic liver abscess; ST, sequence type. Data Sharing Statement The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics Approval This case report was approved by the ethics committee of The Afﬁliated Wuxi Second People’s Hospital of Nanjing Medical University. Consent for Publication Written informed consent for publication of his clinical details and clinical images was obtained from the patient. Author Contributions Bo Zhao and Renjing Hu have contributed equally to this work and they are co-first authors. Conceived the study: Xiaoyun Wang, Bo Zhao; Acquired data, analyzed data: Bo Zhao, Renjing Hu; Performed the microbiological analysis: Renjing Hu; Wrote the manuscript: Bo Zhao; Contributed reagents/materials/analysis tools: Lei Gong, Xiaoyun Wang; and Revised the manuscript: Xiaoyun Wang, Yingwei Zhu, Gaojue Wu. All authors made substantial contributions to the conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. Disclosure The authors declare that they have no conflicts of interest to this work.	UNK (INJECTION)	DrugDosageText	CC BY-NC	33469321	19,979,976	2021
What was the administration route of drug 'HUMAN IMMUNOGLOBULIN G'?	Kawasaki Disease: Predictors of Resistance to Intravenous Immunoglobulin and Cardiac Complications. Kawasaki disease (KD) is the leading cause of acquired cardiac disease in children, in developed countries. To identify predictive factors for resistance to intravenous immunoglobulin (IVIG), calculate the effectiveness of Japanese predictive models and characterize cardiac complications. Retrospective analysis of KD cases admitted in a Portuguese paediatric hospital between january 2006 and july 2018. ROC curves were used to determine predictive factors for resistance and the multivariate logistic regression analysis was used to develop the predictive model. A significance level of 5% was used. 48 patients with a median age of 36 months were included. The IVIG resistance was 21%. Echocardiographic anomalies were noted in 46%, with coronary involvement in 25% of the sample population. As predictive variable of resistance, the C-reactive protein (CRP) presented an AUC ROC = 0.789, optimal cut-off value 15.1 mg/dL, sensitivity (Sn) 77.8% and specificity (Sp) 78.9%. The erythrocyte sedimentation rate (ESR) presented an AUC ROC = 0.781, optimal cut-off value 90.5 mm/h, Sn 66.7% and Sp 85.7%. The model with the two variables showed p = 0.042 and AUC ROC = 0.790. Predictive strength of Japanese models were: Kobayashi (Sn 63.6%, Sp 77.3%), Egami (Sn 66.7%, Sp 73.1%), Sano (Sn 28.6%, Sp 94.1%). CRP and ESR are independent variables that were related to IVIG resistance, with optimal cut-off points of 15.1 mg/dL and 90.5 mm/h, respectively. About half of the patients had some form of cardiac involvement. The Japanese models appeared to be inadequate in our population. (Arq Bras Cardiol. 2021; 116(3):485-491). Introdução A doença de Kawasaki (DK) é uma vasculite aguda e autolimitada que afeta os vasos de média dimensão, sendo a principal causa de cardiopatia adquirida em idade pediátrica.1 A sua etiologia continua incerta, mas vários fatores têm sido associados à doença, nomeadamente genéticos, ambientais e inflamatórios.2 Embora com distribuição mundial, a sua maior prevalência é no Japão, com incidência crescente.3 Em Portugal, um estudo epidemiológico realizado em 2017 descreveu uma incidência anual média de 6,5 por 100.000 crianças com menos de 5 anos de idade.4 Com base nos critérios da American Pediatric Academy de 2004,5 considera-se DK clássica se febre ≥ 5 dias e presença de pelo menos 4 de 5 critérios clínicos adicionais: conjuntivite bilateral não exsudativa, alterações dos lábios e da mucosa oral, exantema polimorfo, alterações das extremidades e linfadenopatia cervical não supurativa. Se febre ≥ 5 dias e apenas 2 ou 3 critérios adicionais, considera-se DK atípica e é necessário recorrer a dados laboratoriais e ecográficos para melhor sustentar o diagnóstico.2 Se não tratada atempadamente, a DK pode complicar-se com aneurismas das artérias coronárias (AAC) em até 25% dos casos.2 Apesar de o envolvimento coronário ser a consequência mais temida da doença, são possíveis outras complicações cardíacas.2,6–8 O tratamento com imunoglobulina intravenosa (IGIV) na fase aguda continua a ser a principal terapêutica e, se administrada nos primeiros 10 dias de doença, diminui a incidência de AAC para 4%.2 A resistência à IGIV ocorre em 10% a 20% dos casos, aumentado a probabilidade de envolvimento coronário.2 Há diferentes abordagens possíveis quando há resistência, nomeadamente uma segunda dose de IGIV, corticosteroides e/ou anticorpos monoclonais.9 Não há descrição de benefício na administração inicial de corticosteroide juntamente com a IGIV a todos os pacientes, sendo atualmente tal terapêutica reservada para os casos refratários.10 Com o objetivo de identificar os casos que potencialmente poderiam ser resistentes ao tratamento com IGIV, e como tal beneficiar de outras terapêuticas na fase inicial, foram desenvolvidos modelos com base em um sistema de escores, nomeadamente os de Kobayashi,11 Egami12 e Sano,13 que foram validados para a população japonesa. No entanto, diversos estudos mostraram que esses modelos são fracos preditores em diversas populações ocidentais.10,14,15 O objetivo desse estudo foi identificar fatores preditores clínicos e analíticos de resistência à IGIV e de envolvimento coronário e construir um modelo preditor de resistência mais adequado para essa população. Como objetivos adicionais, pretendeu-se caracterizar os casos de DK em um hospital pediátrico de nível III nos últimos anos, calcular a eficácia dos escores japoneses nessa amostra e analisar as complicações cardíacas não coronárias da DK. Métodos Amostra Estudo retrospectivo dos casos diagnosticados com DK desde 1º de janeiro de 2006 a 30 de junho de 2018 no Hospital Pediátrico – Centro Hospitalar e Universitário de Coimbra (HP-CHUC). Foram incluídas todas as crianças e adolescentes com idades entre 30 dias e < 18 anos, com diagnóstico e terapêutica de fase aguda realizados no HP-CHUC. Foram excluídos todos os pacientes transferidos para o HP-CHUC já com diagnóstico e/ou terapêutica realizados em outro hospital. Foram utilizados os critérios da American Academy of Pediatrics para diagnóstico de DK clássica e de DK atípica. Considerou-se D1 de febre o dia inaugural de febre, definida como temperatura axilar ≥ 38°C. Considerou-se resistência à IGIV se a febre persistiu 36 horas após a sua administração, tendo sido excluídos todos os casos em que foi administrado corticosteroide concomitantemente com a primeira dose de IGIV. Para classificar o envolvimento coronário, foram usados os escores z (desvios padrões) de Dallaire, definindo-se dilatação se escore z entre 2 e 2,4, aneurisma pequeno se escore z entre 2,5 e 4,9, aneurisma médio se entre 5 e 9,9 e dimensão absoluta inferior a 8 mm, aneurisma gigante se escore z ≥ 10 ou dimensão absoluta ≥ 8 mm. Nos casos em que a informação foi insuficiente para se calcular o escore z, utilizaram-se os valores absolutos, sendo aneurisma pequeno se ≥ 2,5mm e < 4mm, médio se ≥ 4mm e < 8mm e gigante se ≥ 8 mm. Nas complicações cardíacas, os achados ecocardiográficos de hiperecogenicidade e afunilamento das artérias coronárias não foram considerados. Para calcular a eficácia dos modelos, foram excluídos todos os pacientes que não apresentavam os dados necessários para serem considerados como alto ou baixo risco de resistência para determinado modelo. A pontuação e a categorização em pacientes de alto ou baixo risco foram realizadas conforme evidenciado na Tabela 1. Tabela 1 Sistema de pontuação dos modelos avaliados Escore Pontos Alto risco Kobayashi ≥ 4 pontos AST > 100 U/L 2 Na ≤ 133 mmol/L 2 IGIV com febre ≤ D4 2 Neutrófilos/Leucócitos ≥ 80% 2 PC-R ≥ 10 mg/dL 1 Idade ≤ 1 ano 1 Plaquetas ≤ 300.000/uL 1 Egami ≥ 3 pontos ALT ≥ 80 U/L 2 IGIV com febre ≤ D4 1 PC-R ≥ 8 mg/dL 1 Idade ≤ 6 meses 1 Plaquetas ≤ 300.000/uL 1 Sano ≥ 2 pontos AST > 200 U/L 1 Bilirrubina total ≥ 0,9 mg/dL 1 PC-R ≥ 7 mg/dL 1 ALT: alanina aminotransferase; AST: aspartato aminotransferase; dL: decilitro; L: litro; mg: miligrama; IGIV: imunoglobulina intravenosa; mmol: milimole; Na: sódio plasmático; PC-R: proteína C-reativa; U: unidade internacional; uL: microlitro. Análise Estatística Análise estatística efetuada com recurso ao programa SPSS® (IBM®, SPSS® Statistics Inc., Chicago), versão 25.0. Para testar a normalidade da amostra, foi utilizado o teste de Shapiro-Wilk. As variáveis contínuas com distribuição normal foram descritas por meio de média e desvio padrão, as variáveis contínuas sem distribuição normal foram descritas através de mediana e amplitude interquartil (AIQ). Para comparação de variáveis categóricas, foi utilizado o teste exato de Fisher; das variáveis numéricas paramétricas, o teste de t-student não pareado; e das variáveis numéricas não paramétricas, o teste de Mann-Whitney. Foram construídas curvas receiver operating characteristic (ROC) para avaliar a capacidade discriminativa individual de cada variável e identificar pontos de corte para a predição de resistência à IGIV. As variáveis foram consideradas preditoras se com uma area under the curve (AUC) superior a 0,75. Para a elaboração de um modelo preditor de resistência, foi utilizada a regressão logística multivariada. O nível de significância utilizado nesse estudo foi de 5%. Resultados Cumpriram critérios de DK 48 pacientes, sendo 32 (66,7%) do sexo masculino. A mediana de idades foi de 36 meses (AIQ 16,75-89,25), com 62,5% abaixo dos 5 anos de idade e 10,4% acima dos 9 anos. No dia de admissão, a totalidade dos pacientes apresentava-se com febre, com uma mediana de 5 dias de febre (AIQ 4-8), mínimo de 1 e máximo de 14 dias. Dos cinco critérios clínicos principais, observou-se conjuntivite não purulenta em 94%, alterações orais em 90%, exantema em 84%, alterações das extremidades em 75% e linfadenopatia cervical em 69%. Dentre as alterações orais, as mais prevalentes foram a queilite (67%) e o eritema labial (67%), seguidos de eritema da orofaringe (50%) e língua em framboesa (48%). Entre as alterações das extremidades, a mais prevalente foi eritema (52%), seguido de edema duro (31%) e descamação (25%). Os sinais inflamatórios no local de inoculação da vacina Bacillus Calmette-Guérin (BCG) foram descritos em 23%. Foi diagnosticada DK atípica em 17% dos casos. A mediana de dias de internamento foi de 2 dias (AIQ 1-6,75). A totalidade dos pacientes foi medicada em fase aguda com IGIV 2 g/kg com mediana do dia de administração de 6,5 dias (AIQ 5-8). Foram medicados em fase aguda 47 pacientes com ácido acetilsalicílico (AAS) em doses entre 45 a 100mg/kg/dia. Cinco pacientes foram ainda medicados com corticosteroide juntamente com a primeira dose de IGIV. Após a fase aguda, todos os pacientes foram medicados com AAS em dose de 3 a 5 mg/kg/dia, três pacientes com clopidogrel e um com enoxaparina. Verificou-se resistência à IGIV em nove casos (21%), dos quais havia um caso de DK atípica (p = 0,543). Dos nove pacientes resistentes à terapêutica, foi administrada uma segunda dose de IGIV 2 g/kg e a cinco destes, metilprednisolona na dose de 30 mg/kg/dia. Entre as diferentes variáveis avaliadas como preditoras de resistência (Tabela 2), a proteína C-reativa (PC-R) apresentou uma AUC ROC de 0,78 (IC95%: 0,632-0,947) e a velocidade de sedimentação (VS) uma AUC ROC de 0,781 (IC95%: 0,585-0,977). O ponto de corte para a PC-R foi de 15,1 mg/dL com uma sensibilidade (S) de 0,778 e especificidade (E) de 0,789 [Odds ratio (OR) = 13,125 IC95%: 2,271-75,858]. O ponto de corte para a VS foi de 90,5 mm/h, verificando-se uma sensibilidade de 0,667 e especificidade de 0,857 (OR = 12,000 IC95%: 1,718-83,803). Foi ajustado um modelo logístico com as duas variáveis PC-R e VS, modelo este que apresentou um valor p de 0,042 e AUC ROC de 0,790 (IC95%: 0,589-0,992). No ponto de corte ótimo, a sensibilidade foi de 0,833 e a especificidade foi de 0,771. Apresentou ainda uma variância de 25% (Nagelkerke R2 = 0,254). Tabela 2 Análise ROC de diversas variáveis para predizer resistência à IGIV Variável AUC [IC 95%] Idade 0,542 [0,377; 0,708] Dia de administração IGIV 0,595 [0,403; 0,787] Hemoglobina 0,611 [0,416; 0,806] Leucócitos 0,525 [0,331; 0,719] Neutrófilos 0,637 [0,447; 0,828] Plaquetas 0,513 [0,295; 0,732] VS 0,781 [0.585; 0,977] PC-R 0,789 [0,632; 0,947] Na 0,715 [0,475; 0,955] AST 0,648 [0,434; 0,862] ALT 0,693 [0,486; 0,901] Bilirrubina total 0,500 [0,139; 0,861] Albumina 0,693 [0,459; 0,928] ALT: alanina aminotransferase; AST: aspartato aminotransferase; AUC: área abaixo da curva; IC: intervalo de confiança; IGIV: imunoglobulina intravenosa; Na: sódio plasmático; PC-R: proteína C-reativa; ROC: receiver operating curve; VS: velocidade de sedimentação. Ocorreu envolvimento coronário em 12 casos (25%). Sete pacientes desenvolveram dilatação das artérias coronárias e cinco, AAC. Na Tabela 3, comparam-se os grupos sem e com envolvimento coronário. Observaram-se diferenças apenas na duração da febre e na utilização de corticosteroides. O tempo total de febre foi mais elevado no grupo com envolvimento coronário (p = 0,038). Esse grupo recebeu mais vezes corticosteroide (p = 0,009). Quatro desses pacientes já tinham diagnóstico de envolvimento coronário prévio à administração desse fármaco. Dos pacientes com AAC, três cumpriram critérios para aneurismas pequenos, um para aneurismas médios e um para aneurismas gigantes. Esses pacientes estão caracterizados na Tabela 4. Tabela 3 Características dos grupos com e sem envolvimento coronário Variáveis Sem envolvimento coronário (n =36) Envolvimento coronário (n = 12) p Corticosteroide (n = 10) N 4 6 0,009 Resistência à IGIV (n = 9) N 5 4 0,173 Idade (meses) Média ± DP 59,7 ± 57 47,5 ± 30,1 0,35 Dia de resolução da febre Média ± DP 7,4 ± 2,8 9,4 ± 3 0,038 Dia de administração de IGIV Média ± DP 6,5 ± 2,9 7,6 ± 3,5 0,283 DK atípica n 6 2 0,686 Hemoglobina (g/dL) Média ± DP 11,5 ± 1,3 11,2 ± 1,3 0,359 Leucócitos (/uL) Média ± DP 14.174 ± 6.010 15.216 ± 6.918 0,619 Neutrófilos (/uL) Média ± DP 9.515 ± 4.770 10.994 ± 5.629 0,378 Plaquetas (/uL) Média ± DP 32.8389 ± 12.7125 36.2667 ± 28.2652 0,691 PC-R (mg/dL) Média ± DP 10,4 ± 8,8 19,6 ±25 0,243 VS (mm/h) Média ± DP 71,6 ± 19 76,7 ± 33,4 0,672 Na (mmol/L) Média ± DP 137 ± 4 137 ± 5 0,869 AST (U/L) Média ± DP 64 ± 53 101 ± 91 0,222 ALT (U/L) Média ± DP 99 ± 116 113 ± 86 0,712 Bilirrubina total (mg/dL) Média ± DP 1,9 ± 2 2,8 ± 3 0,538 Albumina (g/L) Média ± DP 35,6 ± 4,3 34,9 ± 7,2 0,77 AIQ: amplitude interquartil; ALT: alanina aminotransferase; AST: aspartato aminotransferase; dL: decilitro; DK: doença de Kawasaki; DP: desvio padrão; g: grama; h: hora; IGIV: imunoglobulina intravenosa; L: litro; mg: miligrama; mmol: milimole; n: número absoluto; Na: sódio plasmático; p: valor p; PC-R: proteína C-reativa; U: unidade internacional; uL: microlitro; VS: velocidade de sedimentação. Tabela 4 Características dos pacientes com aneurismas das artérias coronárias Variáveis Paciente 1 Paciente 2 Paciente 3 Paciente 4 Paciente 5 Idade de diagnóstico (meses) 12 60 108 63 4 Sexo masculino Sim Sim Sim Sim Sim Dias de febre 7 6 8 9 14 DK clássico Sim Sim Sim Sim Não Dia de febre na 1ᵃ dose IGIV 7 6 4 6 14 Resistência ao tratamento IGIV Não Não Sim Sim NA Dia de febre na 2ᵃ dose IGIV NA NA 6 8 NA MPDN (30mg/kg/dia) Com 1ᵃ dose IGIV Com 2ᵃ dose IGIV Não NA NA Não NA NA Não Não Sim Sim Não Sim Sim Sim Não Classificação AAC Pequenos Pequenos Pequenos Médios Gigantes Z escore máximo NA 4,46 3,56 6,94 13,81 Artérias atingidas ACD; TC ACD; ACE ACE ACE; ACD ACD; CIR; DAE AAC: aneurismas das artérias coronárias; ACD: artéria coronária direita; ACE: artéria coronária esquerda; CIR: artéria circunflexa; DAE: descendente anterior esquerda; DK: doença de Kawasaki; IGIV: imunoglobulina intravenosa; MPDN: metilprednisolona; NA: não aplicável; TC: tronco comum. Na fase aguda, para além do envolvimento coronário, observou-se derrame pericárdico em 10 casos, insuficiência valvular mitral ligeira em três e disfunção ventricular em três, um dos quais com choque cardiogênico. Um apresentou bloqueio atrioventricular (BAV) de 1ºgrau variável. Após a fase aguda, um paciente manteve envolvimento do sistema de condução e dilatação do ventrículo esquerdo (VE) e um ficou com hipertrofia do VE. Os valores de S, E, valor preditivo positivo (VPP) e valor preditivo negativo (VPN) para os diferentes modelos estão apresentados na tabela 5, sendo apenas incluídos os pacientes a quem foi possível categorizar com alto ou baixo risco. Tabela 5 Sensibilidade, especificidade, valores preditivos positivos e negativos dos diferentes modelos Modelo n S E VPP VPN Kobayasahi 34 63,6% 77,3% 53,8% 81% Egami 39 66,7% 73,1% 50% 82,6% Sano 25 28,6% 94,1% 66,7% 77,3% E: especificidade; n: número de casos incluídos; S: sensibilidade; VPN: valor preditivo negativo; VPP: valor preditivo positivo. Discussão A DK é uma vasculite que, embora não tendo uma incidência tão elevada como no Japão, é, ainda assim uma causa importante de doença em idade pediátrica na nossa população. Um diagnóstico e introdução de terapêutica precoces constituem dois fatores muito importantes para reduzir o risco de envolvimento cardíaco. O presente estudo revelou uma percentagem de casos resistentes à IGIV coincidentes com os 10 a 20% descritos na literatura.2 Ao longo dos anos têm sido desenvolvidos esforços no sentido de encontrar fatores clínicos e laboratoriais que possam prever esta resistência de modo a introduzir mais precocemente terapêuticas coadjuvantes. Existem diversos parâmetros descritos na literatura tais como idade, albumina, transaminases, bilirrubina total, neutrófilos, plaquetas, PC-R, VS, entre outros.14,16–20 Neste estudo observou-se que a PC-R e a VS apresentaram capacidade preditora estatisticamente significativa de resistência à IGIV. No caso da PC-R, o ponto de corte ótimo foi de 15,1 mg/dL, com uma sensibilidade de 0,778, especificidade de 0,789 e um OR de 13,125. Pacientes com PC-R superior a 15,1mg/dL apresentam uma probabilidade de resistência à IGIV cerca de 13 vezes superior aos que têm valores inferiores. Relativamente à VS, o ponto de corte ótimo foi de 90,5 mm/h, com uma sensibilidade de 0,667, especificidade 0,857 e OR 12,000. Pacientes com VS superior a 90,5 mm/h apresentam uma probabilidade de resistência à IGIV cerca de 12 vezes superior aos que têm valores inferiores. Combinando estas duas variáveis independentes obteve-se um modelo estatisticamente significativo (p = 0,042), cujo ponto de corte apresenta uma sensibilidade de 0,833 e especificidade de 0,771. No entanto, a variância explicada pelo modelo é apenas de 25% (Nagelkerke R2 = 0,254), pelo que, embora seja estatisticamente significativo, não pode ser validado, o que se deve em grande parte ao tamanho reduzido da amostra. Todavia, é importante realçar esta tendência no sentido de que as duas variáveis poderão ser importantes para prever a resistência à IGIV, antes da sua infusão. O fato destas variáveis terem sido preditoras de resistência, realça o papel da inflamação exuberante nesta doença, etiologia que é defendida como possível precipitante da resposta imunológica que culmina numa vasculite.21 A etiologia da DK continua incerta, no entanto vários fatores são apontados como predisponentes. Um deles é a imaturidade do sistema imunitário, condição que é apoiada pelo fato de afetar predominantemente crianças com idade inferior a cinco anos. Neste estudo 62,5% dos pacientes pertenciam a esta faixa etária, o que, embora corresponda à maioria da amostra, fica aquém dos 80% descritos na literatura.1 Uma possível explicação para estes resultados é a contribuição genética, sendo as percentagens reportadas baseadas em estudos com variedade étnica alargada, incluindo asiáticos. Verificou-se envolvimento coronário em 25% dos casos, com sete a cumprir critérios de dilatação e cinco de aneurismas das artérias coronárias. Os 10% de incidência de AAC, foram superiores aos 4% reportados para os casos devidamente tratados. Comparando os grupos com e sem envolvimento coronário, verificou-se diferença estatisticamente significativa relativamente ao tempo total de febre (p = 0,038). Este resultado corrobora a ideia da persistência da febre ser deletéria e a necessidade da administração de IGIV preferencialmente até ao 10º dia para evitar sequelas cardíacas.9 O uso de corticosteróide na DK continua a ser tema de debate e controvérsia. O mais consensual é usar metilprednisolona por via intravenosa (MPDN iv) na dose de 15 a 30mg/kg/dia durante três dias.9 A MPDN iv, nos pacientes com DK refratária à IGIV, suprime os níveis de citocinas inflamatórias mais rapidamente que uma 2ᵃ dose de IGIV,9 no entanto não está recomendada como primeira linha. Sleeper et al.10 avaliaram o uso de corticosteróide em diferentes momentos, observando-se diferença estatisticamente significativa no aparecimento de AAC apenas nos pacientes refratários à IGIV e que levaram a administração de segunda dose. Neste estudo também se avaliaram complicações cardíacas e achados ecográficos que não o envolvimento coronário. Das complicações cardíacas em fase aguda mais graves destacaram-se três casos de disfunção ventricular esquerda, um com choque cardiogénico e um BAV de 1º grau, complicações essas também descritas na literatura.2,7 Na fase aguda, foi ainda possível observar 10 pacientes com derrame pericárdico sem compromisso hemodinâmico e três com insuficiência valvular mitral ligeira. Em Paris, Chbeir et al.22 obtiveram relação estatisticamente significativa entre resistência à IGIV, AAC e achados na ecografia cardíaca inicial, tais como derrame pericárdico, hiperecogenicidade das coronárias e dilatação coronária. Os autores desse estudo não consideraram a hiperecogenicidade e o afunilamento das coronárias como fatores relevantes, visto que são achados subjetivos, pouco reprodutíveis e podem ser encontrados tanto em doenças febris como em crianças saudáveis.23 Na fase crônica, um paciente manteve envolvimento do sistema de condução e dilatação do VE e outro ficou com hipertrofia do VE. As repercussões cardíacas a longo prazo na DK estão ainda pouco esclarecidas. Friedman et al.6 mostraram maior ocorrência de efeitos adversos cardíacos a longo prazo, tais como morte, transplante cardíaco, cirurgias de bypass coronário e angioplastia primária em pacientes com DK que desenvolveram AAC com escores z superiores e que foram resistentes à IGIV. Um estudo realizado por Holve et al.8 revelou baixa incidência de efeitos adversos cardíacos até os 21 anos de idade, mas uma maior probabilidade de desenvolver hipertensão arterial após os 15 anos de idade. Os modelos validados no Japão apresentaram fraca utilidade clínica no presente estudo (Tabela 4). O modelo com maior especificidade foi o de Sano, embora com sensibilidade muito baixa e apenas com um pequeno número de casos incluído. O modelo de Egami foi o mais sensível para esta amostra, mas, ainda assim, insuficiente para ser validado. Na base dessa diferença de resultados, pode estar o componente genético. Aliás esses resultados vão ao encontro de outros trabalhos realizados fora do Japão, em que nenhum deles conseguiu validar os modelos nas suas amostras.10,14–17,20 É necessário levar em consideração ainda que existiram algumas diferenças no desenho do estudo relativamente aos modelos japoneses, nomeadamente aplicados apenas a pacientes com DK clássica. Um estudo japonês falhou em validar os modelos em uma amostra de pacientes apenas com DK atípica.24 O modelo de Kobayashi foi validado na população japonesa com uma sensibilidade de 0,86 e especificidade de 0,67.11 Ao contrário do presente estudo, a IGIV foi administrada na dose de 1 g/kg em dois dias consecutivos e foi considerada resistência se febre persistente após 24 horas de início de terapêutica, ou se recrudescência com sintomas após período apirético. O modelo de Egami foi validado com uma sensibilidade de 0,78 e especificidade de 0,76; no entanto, foi definida resistência se ausência de diminuição do valor de PC-R em mais de 50% e persistência da febre 48 horas após administração de IGIV.12 Loomba et al.25 não conseguiram validar o modelo de Egami, mesmo aplicando-o separadamente à DK clássica, atípica e por etnias. O modelo de Sano, validado com uma sensibilidade de 0,77 e especificidade de 0,86, foi o único dos três a ajustar o tamanho dos AAC à superfície corporal.13 No entanto, também usou administração de IGIV na dose de 1 g/kg em 2 dias consecutivos e definiu resistência se persistência de febre após 24 horas do término da terapêutica. Este trabalho apresenta as limitações inerentes ao fato de ser um estudo retrospectivo e com uma amostra de tamanho reduzido. Conclusão A PC-R e a VS são variáveis independentes que mostraram tendência preditora de resistência à IGIV com pontos de corte ótimos de 15,1 mg/dL e 90,5 mm/h, respectivamente. Existe, no entanto, necessidade de um estudo com uma amostra de dimensões adequadas para validar um modelo baseado nesses dois dados analíticos. As complicações cardíacas não se resumem às artérias coronárias, devendo ser mais abrangentes o estudo e o seguimento desses pacientes. Os modelos validados para a população japonesa apresentam utilidade muito limitada na amostra deste estudo, reforçando ainda mais a necessidade e a importância de novas abordagens. Original Article Kawasaki Disease: Predictors of Resistance to Intravenous Immunoglobulin and Cardiac Complications https://orcid.org/0000-0001-7047-4170 Faim Diogo 1 https://orcid.org/0000-0001-7506-0647 Henriques Cláudio 1 Brett Ana 2 Francisco Andreia 1 Rodrigues Fernanda 2 Pires António 1 1 Coimbra Portugal Centro Hospitalar e Universitário de Coimbra EPE - Cardiologia Pediátrica Coimbra - Portugal 2 Coimbra Portugal Centro Hospitalar e Universitário de Coimbra EPE - Urgência e Unidade de Infeciologia, Coimbra - Portugal Mailing Address: Diogo Faim • Centro Hospitalar e Universitário de Coimbra EPE - Praceta, R. Prof. Mota Pinto, 3075 Coimbra 3000-075 – Portugal. E-mail: diogofaim92@gmail.com Abstract Background: Kawasaki disease (KD) is the leading cause of acquired cardiac disease in children, in developed countries. Objectives: To identify predictive factors for resistance to intravenous immunoglobulin (IVIG), calculate the effectiveness of Japanese predictive models and characterize cardiac complications. Methods: Retrospective analysis of KD cases admitted in a Portuguese paediatric hospital between january 2006 and july 2018. ROC curves were used to determine predictive factors for resistance and the multivariate logistic regression analysis was used to develop the predictive model. A significance level of 5% was used. Results: 48 patients with a median age of 36 months were included. The IVIG resistance was 21%. Echocardiographic anomalies were noted in 46%, with coronary involvement in 25% of the sample population. As predictive variable of resistance, the C-reactive protein (CRP) presented an AUC ROC = 0.789, optimal cut-off value 15.1 mg/dL, sensitivity (Sn) 77.8% and specificity (Sp) 78.9%. The erythrocyte sedimentation rate (ESR) presented an AUC ROC = 0.781, optimal cut-off value 90.5 mm/h, Sn 66.7% and Sp 85.7%. The model with the two variables showed p = 0.042 and AUC ROC = 0.790. Predictive strength of Japanese models were: Kobayashi (Sn 63.6%, Sp 77.3%), Egami (Sn 66.7%, Sp 73.1%), Sano (Sn 28.6%, Sp 94.1%). Conclusion: CRP and ESR are independent variables that were related to IVIG resistance, with optimal cut-off points of 15.1 mg/dL and 90.5 mm/h, respectively. About half of the patients had some form of cardiac involvement. The Japanese models appeared to be inadequate in our population. (Arq Bras Cardiol. 2021; 116(3):485-491) Keywords: Kawasaki Disease/complications Mucocutaneous Lymph Node Syndrome/complications Drug Resistance Coronary Artery Disease Immunoglobulin Child Introduction Kawasaki disease (KD) is an acute self-limiting vasculitis, which affects medium-sized vessels and is the leading cause of acquired cardiac disease in pediatric age groups.1 Its etiology remains uncertain, but several factors have been associated to it, namely genetic, environmental, and inflammatory ones.2 Although with a worldwide distribution, its highest prevalence is in Japan, where the incidence is on the rise.3 In Portugal, an epidemiological study carried out in 2017 showed an mean annual incidence of 6.5 per 100,000 children under 5 years of age.4 Based on the 2004 American Pediatric Academy criteria,5 classic KD is considered if fever persists for five days or more and if at least four of five additional clinical criteria are observed: nonexudative bilateral conjunctivitis, alterations of the lips and oral cavity, erythematous rash, changes in the extremities, and cervical lymphadenopathy. If fever lasts for five or more days and only two or three additional criteria are present, it is considered atypical KD, if supported by laboratory and echocardiographic data.2 If not treated within an established period, KD can be complicated by coronary artery aneurysms (CAA) in up to 25% of cases.2 Although coronary artery involvement is the most feared consequence of the disease, other cardiac complications are possible.2,6–8 Treatment with intravenous immunoglobulin (IVIG) in the acute phase administered in the first 10 days of illness reduces the incidence of CAA to 4%.2 IVIG resistance occurs in 10-20% of cases, increasing the likelihood of coronary involvement.2 There are different possible approaches in case of IVIG resistance, such as a second dose of IVIG, corticosteroids and/or monoclonal antibodies.9 No benefit has been described when corticosteroids are used in addition to IVIG in the first instance and this therapeutics is currently reserved for refractory cases.10 In order to identify the cases that could potentially be resistant to treatment with IVIG, and benefit from adjuvant therapies in the initial phase, models based on a scoring system have been developed. Three have been validated in the Japanese population, namely the Kobayashi,11 Egami,12 and Sano scoring systems.13 However, several studies have shown that these models are poor predictors in many western populations.10,14,15 The aim of this study was to identify clinical and laboratory predictive factors regarding resistance to IVIG and coronary artery involvement, and to develop a more suitable predictor model of resistance in this population. Secondary objectives regard characterizing the KD cases admitted to a central pediatric hospital over a period of 13 years, to verify the effectiveness of the Japanese scoring systems in our population sample and to analyze the non-coronary cardiac complications of KD. Methods Sample Retrospective analysis of KD cases admitted to the Hospital Pediátrico – Centro Hospitalar e Universitário de Coimbra (HP-CHUC) diagnosed from 01/01/2006 to 06/30/2018. All patients between 30 days and <18 years with KD and treated with IVIG at diagnosis at HP-CHUC were included in the study. All the patients transferred from outlying centers with a diagnosis of KD and managed at these institutions were excluded. The diagnosis of typical and atypical KD was based on the American Academy of Pediatrics criteria. We considered day one of fever on the day the fever started, defined as axillar temperature ≥ 38ºC. Resistance to IVIG was considered if the fever persisted 36 hours after its administration. All patients who received corticosteroids simultaneously with the first dose of IVIG were excluded from the resistance quantification. Dallaire z scores were used to classify the coronary artery morphology defining: coronary artery ectasia if z score between 2 and 2.4, small aneurysm if z score between 2.5 and 4.9, medium aneurysm if z score between 5 and 9.9 and absolute dimension < 8 mm, and giant aneurysm if z score ≥ 10 or absolute dimension ≥ 8 mm. If unable to calculate the z score, the absolute dimensions were used, being small aneurysm if ≥ 2.5 mm and < 4mm, medium aneurysm if ≥ 4 mm and < 8 mm and giant aneurysm if ≥ 8 mm. Regarding other cardiac complications, the coronary artery hyperechogenicity and lack of tapering on echocardiography were not considered as echocardiographic diagnostic criteria. To calculate the effectiveness of the Japanese models, all patients who did not have the necessary data to be considered as high or low risk of IVIG resistance were excluded. Scoring and categorization in high or low risk patients were performed as shown in Table 1. Table 1 Scoring system of the Japanese modelss Model Score High risk Kobayashi ≥ 4 points AST > 100 U/L 2 Na ≤ 133 mmol/L 2 IVIG with fever ≤ 4 days 2 Neutrophils/Leucocytes ≥ 80% 2 CRP ≥ 10 mg/dL 1 Age ≤ 1 year old 1 Platelets ≤ 300,000/µL 1 Egami ≥ 3 points ALT ≥ 80 U/L 2 IVIG with fever ≤ 4 days 1 CRP ≥ 8 mg/dL 1 Age ≤ 6 months 1 Platelets ≤ 300,000/µL 1 Sano ≥ 2 points AST > 200 U/L 1 Total bilirubin ≥ 0.9 mg/dL 1 CRP ≥ 7 mg/dL 1 AST: aspartate transaminase; U: international unity; L: liter; Na: serum sodium; mmol: millimole; IVIG: intravenous immunoglobulin; CRP: C-reactive protein; mg: milligram; dL: deciliter; µL: microliter; ALT: alanine transaminase. Statistical Analysis The SPSS® (IBM®, SPSS® Statistics Inc., Chicago) program version 25.0 was used the performed the statistical analysis. The Shapiro-Wilk test was used to test the normality of the variables. The continuous variables with normal distribution were described using mean and standard deviation (SD) and continuous variables without normal distribution were described using median and interquartile range (IQR). We used the Fisher’s exact test to compare categorical variables, the Student’s t-test to compare parametric variables and the Mann-Whitney test to compare the non-parametric ones. The Receiver Operating Characteristic (ROC) curves were used to evaluate the individual discriminative capacity of each variable and to identify the optimal cutoff points to predict resistance to IVIG. The variables were considered as good predictors if the area under the curve (AUC) > 0.75. Multivariate logistic regression analysis was used to develop the predictive resistance model. A significance level of 5% was used. Results Forty-eight patients met the KD criteria, of whom 32 (66.7%) were male. The median age was 36 months (IQR 16.75-89.25), 62.5% of patients were less than five years old and 10.4% over nine years old. On the day of admission, all the patients presented with fever, with a median duration of five days (IQR 4-8), minimum of one day and maximum of 14 days. Among the five main clinical criteria, nonexudative bilateral conjunctivitis was observed in 94% of cases, alterations of the lips and oral cavity in 90%, erythematous rash in 84%, changes in extremities in 75%, and cervical lymphadenopathy in 69%. The most common findings among oral alterations were cheilitis (67%) and lip erythema (67%), followed by erythema of the oropharynx (50%) and the strawberry tongue (48%). The most prevalent changes in the extremities were erythema (52%), followed by swelling (31%) and peeling (25%). Inflammatory signs at the Bacillus Calmette-Guérin (BCG) vaccination site were observed in 23% of patients. Atypical KD was diagnosed in 17% of the cases. The median duration of hospital stay was two days (IQR 1-6.75). During the acute phase, IVIG 2g/kg was administered to all patients. The median day of illness of the administration was 6.5 (IQR 5-8). Simultaneously, 45-100mg/kg of acetylsalicylic acid (ASA) were administered to 47 patients in the acute phase. Five children received corticosteroids together with the first dose of IVIG. After the acute phase, all patients were medicated with ASA 3-5 mg/kg/day, three patients with clopidogrel and one with enoxaparin. Nine patients were resistant to the IVIG (21%), of which one had atypical KD (p = 0.543). All nine repeated IVIG administration, five of which with methylprednisolone 30 mg/kg/day. Among the variables evaluated as predictors of IVIG resistance (Table 2), C-reactive protein (CRP) presented an AUC ROC of 0.78 (95% confidence interval (CI): 0.632 – 0.947), and the erythrocyte sedimentation rate (ESR), an AUC ROC of 0.781 (95%CI: 0.585 – 0.977). The optimal cut-off value for CRP was 15.1 mg/dL with sensitivity (Sn) of 77.8% and specificity (Sp) of 78.9% (Odds ratio (OR) = 13.125 95%CI: 2.271 – 75.858). The optimal cut-off value for ESR was 90.5 mm/h, with Sn of 66.7% and Sp of 85.7% (OR = 12.000 95%CI: 1.718 – 83.803). A logistic model was developed with these two variables, with a p-value of 0.042, AUC ROC of 0.79 (95%CI: 0.589 – 0.992), with Sn of 83.3% and Sp of 77.1%, but with a 25% variance (Nagelkerke R2 = 0.254). Table 2 Receiver operating characteristic analysis of several variables to predict resistance to intravenous hemoglobulin Characteristic AUC [95%CI] Age 0.542 [0.377; 0.708] IVIG administration day 0.595 [0.403; 0.787] Hemoglobin 0.611 [0.416; 0.806] Leucocytes 0.525 [0.331; 0.719] Neutrophils 0.637 [0.447; 0.828] Platelets 0.513 [0.295; 0.732] ESR 0.781 [0.585; 0.977] CRP 0.789 [0.632; 0.947] Na 0.715 [0.475; 0.955] AST 0.648 [0.434; 0.862] ALT 0.693 [0.486; 0.901] Total bilirubin 0.500 [0,139; 0.861] Albumin 0.693 [0.459; 0.928] AUC: area under the curve; CI: confidence interval; IVIG: intravenous immunoglobulin; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; Na: serum sodium; AST: aspartate transaminase; ALT: alanine transaminase. Coronary artery changes were found in 12 children (25%), seven with ectasia and five with CAA. The comparison between groups with and without coronary artery involvement is shown in Table 3. The duration of fever and the use of corticosteroids were the only significant differences between these two groups. Patients with coronary artery involvement had longer duration of fever (p = 0.038) and greater need for corticotherapy (p = 0.009). Four patients had CAA when methylprednisolone was started. Among the five patients with CAA, three met the criteria for small aneurysms, one for medium aneurysms and one for giant ones. These patients are summarized in Table 4. Table 3 Characteristics of groups with and without coronary artery involvement Characteristics Without coronary involvement (n =36) Coronary involvement (n= 12) p - value Corticoid (n=10) n 4 6 0.009 Resistance to IVIG (n=9) n 5 4 0.173 Age (months) Mean ± sd 59.7 ± 57 47.5 ± 30.1 0.35 Fever duration (days) Mean ± sd 7.4 ± 2.8 9.4 ± 3 0.038 IVIG administration day Mean ± sd 6.5 ± 2.9 7.6 ± 3.5 0.283 Atypical KD n 6 2 0.686 Haemoglobin (g/dL) Mean ± sd 11.5 ± 1.3 11.2 ± 1.3 0.359 Leucocytes (/µL) Mean ± sd 14,174 ± 6,010 15,216 ± 6,918 0.619 Neutrophils (/µL) Mean ± sd 9,515 ± 4,770 10,994 ± 5,629 0.378 Platelets (/µL) Mean ± sd 328,389 ± 127,125 362,667 ± 282,652 0.691 CRP (mg/dL) Mean ± sd 10.4 ± 8.8 19.6 ±25 0.243 ESR (mm/h) Mean ± sd 71.6 ± 19 76.7 ± 33.4 0.672 Na (mmol/L) Mean ± sd 137 ± 4 137 ± 5 0.869 AST (U/L) Mean ± sd 64 ± 53 101 ± 91 0.222 ALT (U/L) Mean ± sd 99 ± 116 113 ± 86 0.712 Total bilirubin (mg/dL) Mean ± sd 1.9 ± 2 2.8 ± 3 0.538 Albumin (g/L) Mean ± sd 35.6 ± 4.3 34.9 ± 7.2 0.77 n: absolute value; IVIG: intravenous immunoglobulin; sd: standard deviation; KD: Kawasaki disease; g: gram; dL: deciliter; µL: microliter; CRP: C-reactive protein; mg: milligram; ESR: erythrocyte sedimentation rate; h: hour; Na: serum sodium; mmol: millimole; L: litre; AST: aspartate transaminase; U: international unity; ALT: alanine transaminase. Table 4 Characteristics of patients with coronary artery aneurysms Characteristics Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Age (months) 12 60 108 63 4 Male Yes Yes Yes Yes Yes Fever duration (days) 7 6 8 9 14 Classic KD Yes Yes Yes Yes No Day of fever in IVIG 1st dose 7 6 4 6 14 Resistance to IVIG No No Yes Yes NA Day of fever in IVIG 2nd dose NA NA 6 8 NA MPDN (30mg/Kg/day) With IVIG 1st dose With IVIG 2nd dose No NA NA No NA NA Yes No Yes Yes No Yes Yes Yes No CAA classification Small Small Small Medium Giant Maximum z score NA 4.46 3.56 6.94 13.81 Arteries involved RCA; CT RCA; LCA LCA RCA; LCA RCA; LAD; LCX KD: Kawasaki disease; IVIG: intravenous immunoglobulin; MPDN: methylprednisolone; mg: milligram; Kg: kilogram; CAA: coronary artery aneurysms; RCA: right coronary artery, CT: common trunk; LCA: left coronary artery; LAD: left anterior descending artery; NA: not applicable; LCX: left circumflex artery. In the acute phase, in addition to coronary involvement, 10 patients presented with pericardial effusion, three with mild mitral valve regurgitation, two with left ventricular systolic dysfunction, one with cardiogenic shock and one with variable first degree atrioventricular (AV) block. After the acute phase, the patient with the AV block developed left ventricle (LV) dilation and another patient developed LV hypertrophy. Table 5 summarizes Sn, Sp,and the positive (PPV) and negative predictive values (NPV) for the Japanese models in our sample. Table 5 Statistical values of the Japanese models in our study Model n Sn (%) Sp (%) PPV (%) NPV (%) Kobayasahi 34 63.6 77.3 53.8 81 Egami 39 66.7 73.1 50 82.6 Sano 25 28.6 94.1 66.7 77.3 n: absolute value; Sn: sensitivity; Sp: Specificity; PPV: positive predictive value; NPV: negative predictive value. Discussion Despite the lower incidence compared to Japan, KD is a vasculitis that is still an important cause of pediatric disease in our population. Early diagnosis and management are two important factors that appear to reduce cardiac involvement. Our study revealed an incidence of IVIG resistance similar to the 10 to 20% described in the literature.2 Over the years, efforts have been made to find clinical and laboratory factors that can predict this resistance in order to introduce adjuvant therapies at an early stage of the disease. There are several parameters in the literature that have been studied for this purpose, such as age, serum albumin, transaminases, total bilirubin, neutrophils count, platelet count, CRP, ESR, among others.14,16–20 In our study, CRP and ESR presented a statically significant predictive capacity in relation to IVIG resistance. For CRP, the optimal cut-off point was 15.1 mg/dL (Sn 77.8%, Sp 78.9%, OR 13.125). Patients with CRP values above 15.1 mg/dL are about 13 times more likely to be resistant to IVIG than those with lower values. Concerning ESR, the optimal cut-off point was 90.5 mm/h (Sn 66.7%, Sp 85.7%, OR 12.000). Patients with ESR greater than 90.5 mm/h have a probability of resistance to IVIG approximately 12 times higher than those with lower values. Combining these two independent variables, a statistically significant model was obtained (p = 0.042), whose cut-off point has Sn of 83.3% and Sp of 77.1%. Despite these encouraging results, the variance explained by the model is only 25% (Nagelkerke R2 = 0.254). Thus, although statistically significant, it cannot be validated, which is largely due to the small sample size. Nevertheless, based on these trends, the base-line values for CRP and ESR should be known prior to IVIG administration. The resistance predictor capacity highlights the role of inflammation in this disease, a possible underlying trigger in KD vasculitis.21 The etiology of KD remains uncertain, however, predisposing factors have been put forward. One, is the immaturity of the immune system, a theory that is supported by the fact that KD predominantly affects children under the age of five. In our study, 62.5% of the patients belonged to this age group, which, although corresponding to the majority of the sample, is below the 80% described in the literature.1 A possible explanation for this result is genetic contribution, since the incidences described in the literature are from studies with a wide range of ethnicities, including Asian children. Coronary artery involvement occurred in 12 (25%) children, seven with ectasia and five with CAA. Therefore, the incidence of CAA was 10%, which is higher than the 4% reported in the literature. Comparing the groups with and without coronary artery involvement, a statistically significant difference was found regarding the duration of fever (p = 0.038). This result highlights the deleterious effects of persistent fever and the need for IVIG administration, preferably up to the tenth day of the disease, in order to avoid cardiac sequelae.9 The use of corticosteroids in KD is still a topic of debate and controversy. The most consensual is the use intravenous methylprednisolone (MPDN) at a dose of 15 to 30 mg/kg/day, for three days.9 In patients with refractory KD, MPDN supresses the inflammatory cytokine levels more quickly than a second dose of IVIG,9 although it is not recommended as a first-line treatment. Sleeper et al.10 evaluated the impact of corticosteroids at different times of the disease and showed that, with regards to the development of CAA, the only statistically significant difference was in those refractory to the first dose of IVIG which combined corticosteroids with the second dose of IVIG. The cardiac complications and echocardiographic findings, others than coronary artery involvement were also evaluated. Three cases of left ventricular systolic dysfunction were identified, one of which with cardiogenic shock, a complication also described in the literature.2,7 In the acute phase, ten patients presented with pericardial effusion without hemodynamic compromise, three patients with mild mitral valve regurgitation and one with first degree AV block. Chbeir et al.22 found a relation between resistance to IVIG, CAA, and initial cardiac echocardiographic findings such as pericardial effusion, coronary hyperechogenicity, and coronary ectasia. The coronary hyperechogenicity and the lack of tapering on echocardiography were not considered as relevant factors, since they are subjective findings, poorly reproducible and can be found both in febrile illnesses and in healthy children.23 During the chronic phase, one patient remained with conduction system impairment and developed LV dilation, and another patient developed LV hypertrophy. The long-term cardiac repercussions in KD remain unclear. Friedman et al.6 reported an increase in the occurrence of long-term adverse cardiac effects, leading to primary angioplasty, coronary bypass surgery, heart transplantation, and death, in patients who developed CAA with higher z scores and who were initially resistant to IVIG. A study by Holve et al.8 revealed a low incidence of adverse cardiac effects in subjects up to 21 years of age, but a greater likelihood of developing high blood pressure from the age of 15. Japanese predictive models presented poor clinical utility in this study (Table 4). The model with the highest specificity was Sano’s, although with very low sensitivity and with only a small number of cases included. Egami’s model was the most sensitive, however, not powerful enough to be validated. The genetic component may be the explanation for these differences. In fact, the results presented here are similar to those of other studies carried out outside Japan, in which none were able to validate the models in their samples.10,14–17,20 It is still important to note the differences in the study design in relation to the Japanese models, namely that they were applied only to patients with classical KD. Another Japanese study failed to validate the models in a sample exclusively composed of atypical KD cases.24 The Kobayashi model was validated for the Japanese population with Sn of 86% and Sp of 67%.11 Contrary to the present study, IVIG was administered at a dose of 1 g/kg on two consecutive days and resistance was considered if fever persisted 24 hours after the beginning of the treatment, or in case of recurrence after a period without fever. The Egami model was validated with Sn of 78% and Sp of 76%, however, resistance was considered if the CRP value did not decrease by more than 50% and fever persisted for longer than 48 hours after IVIG administration.12 Loomba et al.25 were not able to validate the Egami model even when applying it separately to classical and atypical KD, and by ethnicity. The Sano model, validated with Sn of 77% and Sp of 86%, was the only one of the three to adjust the size of the CAA to the body surface.13 However, it also used IVIG at a dose of 1 g/kg on two consecutive days and defined resistance if fever persisted 24 hours after the end of therapy. The main limitations of this analysis are related to its retrospective methodology and sample size. Conclusions CRP and ESR are independent variables that showed a predictive trend regarding resistance to IVIG, with optimal cut-off values of 15.1 mg/dL and 90.5 mm/h, respectively. However, there is a need for a multicenter study with a sample of adequate dimensions to validate a model based on these two analytical parameters. Cardiac complications are not limited to coronary arteries, and the study and follow-up of these patients should be more widespread. The validated models for the Japanese population have very limited utility in our population, further reinforcing the need and importance of new approaches. Fontes de Financiamento O presente estudo não contou com fontes de financiamento externas. Vinculação Acadêmica Não há vinculação deste estudo a programas de pós-graduação. Aprovação Ética e Consentimento Informado Este artigo não contém estudos com humanos ou animais realizados por nenhum dos autores. Author Contributions Conception and design of the research: Faim D, Henriques C, Brett A, Francisco A, Rodrigues F, Pires A; Data acquisition: Faim D, Henriques C; Analysis and interpretation of the data: Faim D, Brett A, Francisco A; Statistical analysis and Writing of the manuscript: Faim D; Critical revision of the manuscript for intellectual content: Faim D, Brett A, Francisco A, Rodrigues F, Pires A. Potential Conflict of Interest The authors report no conflict of interest concerning the materials and methods used in this study or the findings specified in this paper. Sources of Funding There was no external funding source for this study. Study Association This study is not associated with any thesis or dissertation. Ethics Approval and Consent to Participate This article does not contain any studies with human participants or animals performed by any of the authors.	Intravenous (not otherwise specified)	DrugAdministrationRoute	CC BY	33470332	19,012,502	2021-03
What was the dosage of drug 'ASPIRIN'?	Kawasaki Disease: Predictors of Resistance to Intravenous Immunoglobulin and Cardiac Complications. Kawasaki disease (KD) is the leading cause of acquired cardiac disease in children, in developed countries. To identify predictive factors for resistance to intravenous immunoglobulin (IVIG), calculate the effectiveness of Japanese predictive models and characterize cardiac complications. Retrospective analysis of KD cases admitted in a Portuguese paediatric hospital between january 2006 and july 2018. ROC curves were used to determine predictive factors for resistance and the multivariate logistic regression analysis was used to develop the predictive model. A significance level of 5% was used. 48 patients with a median age of 36 months were included. The IVIG resistance was 21%. Echocardiographic anomalies were noted in 46%, with coronary involvement in 25% of the sample population. As predictive variable of resistance, the C-reactive protein (CRP) presented an AUC ROC = 0.789, optimal cut-off value 15.1 mg/dL, sensitivity (Sn) 77.8% and specificity (Sp) 78.9%. The erythrocyte sedimentation rate (ESR) presented an AUC ROC = 0.781, optimal cut-off value 90.5 mm/h, Sn 66.7% and Sp 85.7%. The model with the two variables showed p = 0.042 and AUC ROC = 0.790. Predictive strength of Japanese models were: Kobayashi (Sn 63.6%, Sp 77.3%), Egami (Sn 66.7%, Sp 73.1%), Sano (Sn 28.6%, Sp 94.1%). CRP and ESR are independent variables that were related to IVIG resistance, with optimal cut-off points of 15.1 mg/dL and 90.5 mm/h, respectively. About half of the patients had some form of cardiac involvement. The Japanese models appeared to be inadequate in our population. (Arq Bras Cardiol. 2021; 116(3):485-491). Introdução A doença de Kawasaki (DK) é uma vasculite aguda e autolimitada que afeta os vasos de média dimensão, sendo a principal causa de cardiopatia adquirida em idade pediátrica.1 A sua etiologia continua incerta, mas vários fatores têm sido associados à doença, nomeadamente genéticos, ambientais e inflamatórios.2 Embora com distribuição mundial, a sua maior prevalência é no Japão, com incidência crescente.3 Em Portugal, um estudo epidemiológico realizado em 2017 descreveu uma incidência anual média de 6,5 por 100.000 crianças com menos de 5 anos de idade.4 Com base nos critérios da American Pediatric Academy de 2004,5 considera-se DK clássica se febre ≥ 5 dias e presença de pelo menos 4 de 5 critérios clínicos adicionais: conjuntivite bilateral não exsudativa, alterações dos lábios e da mucosa oral, exantema polimorfo, alterações das extremidades e linfadenopatia cervical não supurativa. Se febre ≥ 5 dias e apenas 2 ou 3 critérios adicionais, considera-se DK atípica e é necessário recorrer a dados laboratoriais e ecográficos para melhor sustentar o diagnóstico.2 Se não tratada atempadamente, a DK pode complicar-se com aneurismas das artérias coronárias (AAC) em até 25% dos casos.2 Apesar de o envolvimento coronário ser a consequência mais temida da doença, são possíveis outras complicações cardíacas.2,6–8 O tratamento com imunoglobulina intravenosa (IGIV) na fase aguda continua a ser a principal terapêutica e, se administrada nos primeiros 10 dias de doença, diminui a incidência de AAC para 4%.2 A resistência à IGIV ocorre em 10% a 20% dos casos, aumentado a probabilidade de envolvimento coronário.2 Há diferentes abordagens possíveis quando há resistência, nomeadamente uma segunda dose de IGIV, corticosteroides e/ou anticorpos monoclonais.9 Não há descrição de benefício na administração inicial de corticosteroide juntamente com a IGIV a todos os pacientes, sendo atualmente tal terapêutica reservada para os casos refratários.10 Com o objetivo de identificar os casos que potencialmente poderiam ser resistentes ao tratamento com IGIV, e como tal beneficiar de outras terapêuticas na fase inicial, foram desenvolvidos modelos com base em um sistema de escores, nomeadamente os de Kobayashi,11 Egami12 e Sano,13 que foram validados para a população japonesa. No entanto, diversos estudos mostraram que esses modelos são fracos preditores em diversas populações ocidentais.10,14,15 O objetivo desse estudo foi identificar fatores preditores clínicos e analíticos de resistência à IGIV e de envolvimento coronário e construir um modelo preditor de resistência mais adequado para essa população. Como objetivos adicionais, pretendeu-se caracterizar os casos de DK em um hospital pediátrico de nível III nos últimos anos, calcular a eficácia dos escores japoneses nessa amostra e analisar as complicações cardíacas não coronárias da DK. Métodos Amostra Estudo retrospectivo dos casos diagnosticados com DK desde 1º de janeiro de 2006 a 30 de junho de 2018 no Hospital Pediátrico – Centro Hospitalar e Universitário de Coimbra (HP-CHUC). Foram incluídas todas as crianças e adolescentes com idades entre 30 dias e < 18 anos, com diagnóstico e terapêutica de fase aguda realizados no HP-CHUC. Foram excluídos todos os pacientes transferidos para o HP-CHUC já com diagnóstico e/ou terapêutica realizados em outro hospital. Foram utilizados os critérios da American Academy of Pediatrics para diagnóstico de DK clássica e de DK atípica. Considerou-se D1 de febre o dia inaugural de febre, definida como temperatura axilar ≥ 38°C. Considerou-se resistência à IGIV se a febre persistiu 36 horas após a sua administração, tendo sido excluídos todos os casos em que foi administrado corticosteroide concomitantemente com a primeira dose de IGIV. Para classificar o envolvimento coronário, foram usados os escores z (desvios padrões) de Dallaire, definindo-se dilatação se escore z entre 2 e 2,4, aneurisma pequeno se escore z entre 2,5 e 4,9, aneurisma médio se entre 5 e 9,9 e dimensão absoluta inferior a 8 mm, aneurisma gigante se escore z ≥ 10 ou dimensão absoluta ≥ 8 mm. Nos casos em que a informação foi insuficiente para se calcular o escore z, utilizaram-se os valores absolutos, sendo aneurisma pequeno se ≥ 2,5mm e < 4mm, médio se ≥ 4mm e < 8mm e gigante se ≥ 8 mm. Nas complicações cardíacas, os achados ecocardiográficos de hiperecogenicidade e afunilamento das artérias coronárias não foram considerados. Para calcular a eficácia dos modelos, foram excluídos todos os pacientes que não apresentavam os dados necessários para serem considerados como alto ou baixo risco de resistência para determinado modelo. A pontuação e a categorização em pacientes de alto ou baixo risco foram realizadas conforme evidenciado na Tabela 1. Tabela 1 Sistema de pontuação dos modelos avaliados Escore Pontos Alto risco Kobayashi ≥ 4 pontos AST > 100 U/L 2 Na ≤ 133 mmol/L 2 IGIV com febre ≤ D4 2 Neutrófilos/Leucócitos ≥ 80% 2 PC-R ≥ 10 mg/dL 1 Idade ≤ 1 ano 1 Plaquetas ≤ 300.000/uL 1 Egami ≥ 3 pontos ALT ≥ 80 U/L 2 IGIV com febre ≤ D4 1 PC-R ≥ 8 mg/dL 1 Idade ≤ 6 meses 1 Plaquetas ≤ 300.000/uL 1 Sano ≥ 2 pontos AST > 200 U/L 1 Bilirrubina total ≥ 0,9 mg/dL 1 PC-R ≥ 7 mg/dL 1 ALT: alanina aminotransferase; AST: aspartato aminotransferase; dL: decilitro; L: litro; mg: miligrama; IGIV: imunoglobulina intravenosa; mmol: milimole; Na: sódio plasmático; PC-R: proteína C-reativa; U: unidade internacional; uL: microlitro. Análise Estatística Análise estatística efetuada com recurso ao programa SPSS® (IBM®, SPSS® Statistics Inc., Chicago), versão 25.0. Para testar a normalidade da amostra, foi utilizado o teste de Shapiro-Wilk. As variáveis contínuas com distribuição normal foram descritas por meio de média e desvio padrão, as variáveis contínuas sem distribuição normal foram descritas através de mediana e amplitude interquartil (AIQ). Para comparação de variáveis categóricas, foi utilizado o teste exato de Fisher; das variáveis numéricas paramétricas, o teste de t-student não pareado; e das variáveis numéricas não paramétricas, o teste de Mann-Whitney. Foram construídas curvas receiver operating characteristic (ROC) para avaliar a capacidade discriminativa individual de cada variável e identificar pontos de corte para a predição de resistência à IGIV. As variáveis foram consideradas preditoras se com uma area under the curve (AUC) superior a 0,75. Para a elaboração de um modelo preditor de resistência, foi utilizada a regressão logística multivariada. O nível de significância utilizado nesse estudo foi de 5%. Resultados Cumpriram critérios de DK 48 pacientes, sendo 32 (66,7%) do sexo masculino. A mediana de idades foi de 36 meses (AIQ 16,75-89,25), com 62,5% abaixo dos 5 anos de idade e 10,4% acima dos 9 anos. No dia de admissão, a totalidade dos pacientes apresentava-se com febre, com uma mediana de 5 dias de febre (AIQ 4-8), mínimo de 1 e máximo de 14 dias. Dos cinco critérios clínicos principais, observou-se conjuntivite não purulenta em 94%, alterações orais em 90%, exantema em 84%, alterações das extremidades em 75% e linfadenopatia cervical em 69%. Dentre as alterações orais, as mais prevalentes foram a queilite (67%) e o eritema labial (67%), seguidos de eritema da orofaringe (50%) e língua em framboesa (48%). Entre as alterações das extremidades, a mais prevalente foi eritema (52%), seguido de edema duro (31%) e descamação (25%). Os sinais inflamatórios no local de inoculação da vacina Bacillus Calmette-Guérin (BCG) foram descritos em 23%. Foi diagnosticada DK atípica em 17% dos casos. A mediana de dias de internamento foi de 2 dias (AIQ 1-6,75). A totalidade dos pacientes foi medicada em fase aguda com IGIV 2 g/kg com mediana do dia de administração de 6,5 dias (AIQ 5-8). Foram medicados em fase aguda 47 pacientes com ácido acetilsalicílico (AAS) em doses entre 45 a 100mg/kg/dia. Cinco pacientes foram ainda medicados com corticosteroide juntamente com a primeira dose de IGIV. Após a fase aguda, todos os pacientes foram medicados com AAS em dose de 3 a 5 mg/kg/dia, três pacientes com clopidogrel e um com enoxaparina. Verificou-se resistência à IGIV em nove casos (21%), dos quais havia um caso de DK atípica (p = 0,543). Dos nove pacientes resistentes à terapêutica, foi administrada uma segunda dose de IGIV 2 g/kg e a cinco destes, metilprednisolona na dose de 30 mg/kg/dia. Entre as diferentes variáveis avaliadas como preditoras de resistência (Tabela 2), a proteína C-reativa (PC-R) apresentou uma AUC ROC de 0,78 (IC95%: 0,632-0,947) e a velocidade de sedimentação (VS) uma AUC ROC de 0,781 (IC95%: 0,585-0,977). O ponto de corte para a PC-R foi de 15,1 mg/dL com uma sensibilidade (S) de 0,778 e especificidade (E) de 0,789 [Odds ratio (OR) = 13,125 IC95%: 2,271-75,858]. O ponto de corte para a VS foi de 90,5 mm/h, verificando-se uma sensibilidade de 0,667 e especificidade de 0,857 (OR = 12,000 IC95%: 1,718-83,803). Foi ajustado um modelo logístico com as duas variáveis PC-R e VS, modelo este que apresentou um valor p de 0,042 e AUC ROC de 0,790 (IC95%: 0,589-0,992). No ponto de corte ótimo, a sensibilidade foi de 0,833 e a especificidade foi de 0,771. Apresentou ainda uma variância de 25% (Nagelkerke R2 = 0,254). Tabela 2 Análise ROC de diversas variáveis para predizer resistência à IGIV Variável AUC [IC 95%] Idade 0,542 [0,377; 0,708] Dia de administração IGIV 0,595 [0,403; 0,787] Hemoglobina 0,611 [0,416; 0,806] Leucócitos 0,525 [0,331; 0,719] Neutrófilos 0,637 [0,447; 0,828] Plaquetas 0,513 [0,295; 0,732] VS 0,781 [0.585; 0,977] PC-R 0,789 [0,632; 0,947] Na 0,715 [0,475; 0,955] AST 0,648 [0,434; 0,862] ALT 0,693 [0,486; 0,901] Bilirrubina total 0,500 [0,139; 0,861] Albumina 0,693 [0,459; 0,928] ALT: alanina aminotransferase; AST: aspartato aminotransferase; AUC: área abaixo da curva; IC: intervalo de confiança; IGIV: imunoglobulina intravenosa; Na: sódio plasmático; PC-R: proteína C-reativa; ROC: receiver operating curve; VS: velocidade de sedimentação. Ocorreu envolvimento coronário em 12 casos (25%). Sete pacientes desenvolveram dilatação das artérias coronárias e cinco, AAC. Na Tabela 3, comparam-se os grupos sem e com envolvimento coronário. Observaram-se diferenças apenas na duração da febre e na utilização de corticosteroides. O tempo total de febre foi mais elevado no grupo com envolvimento coronário (p = 0,038). Esse grupo recebeu mais vezes corticosteroide (p = 0,009). Quatro desses pacientes já tinham diagnóstico de envolvimento coronário prévio à administração desse fármaco. Dos pacientes com AAC, três cumpriram critérios para aneurismas pequenos, um para aneurismas médios e um para aneurismas gigantes. Esses pacientes estão caracterizados na Tabela 4. Tabela 3 Características dos grupos com e sem envolvimento coronário Variáveis Sem envolvimento coronário (n =36) Envolvimento coronário (n = 12) p Corticosteroide (n = 10) N 4 6 0,009 Resistência à IGIV (n = 9) N 5 4 0,173 Idade (meses) Média ± DP 59,7 ± 57 47,5 ± 30,1 0,35 Dia de resolução da febre Média ± DP 7,4 ± 2,8 9,4 ± 3 0,038 Dia de administração de IGIV Média ± DP 6,5 ± 2,9 7,6 ± 3,5 0,283 DK atípica n 6 2 0,686 Hemoglobina (g/dL) Média ± DP 11,5 ± 1,3 11,2 ± 1,3 0,359 Leucócitos (/uL) Média ± DP 14.174 ± 6.010 15.216 ± 6.918 0,619 Neutrófilos (/uL) Média ± DP 9.515 ± 4.770 10.994 ± 5.629 0,378 Plaquetas (/uL) Média ± DP 32.8389 ± 12.7125 36.2667 ± 28.2652 0,691 PC-R (mg/dL) Média ± DP 10,4 ± 8,8 19,6 ±25 0,243 VS (mm/h) Média ± DP 71,6 ± 19 76,7 ± 33,4 0,672 Na (mmol/L) Média ± DP 137 ± 4 137 ± 5 0,869 AST (U/L) Média ± DP 64 ± 53 101 ± 91 0,222 ALT (U/L) Média ± DP 99 ± 116 113 ± 86 0,712 Bilirrubina total (mg/dL) Média ± DP 1,9 ± 2 2,8 ± 3 0,538 Albumina (g/L) Média ± DP 35,6 ± 4,3 34,9 ± 7,2 0,77 AIQ: amplitude interquartil; ALT: alanina aminotransferase; AST: aspartato aminotransferase; dL: decilitro; DK: doença de Kawasaki; DP: desvio padrão; g: grama; h: hora; IGIV: imunoglobulina intravenosa; L: litro; mg: miligrama; mmol: milimole; n: número absoluto; Na: sódio plasmático; p: valor p; PC-R: proteína C-reativa; U: unidade internacional; uL: microlitro; VS: velocidade de sedimentação. Tabela 4 Características dos pacientes com aneurismas das artérias coronárias Variáveis Paciente 1 Paciente 2 Paciente 3 Paciente 4 Paciente 5 Idade de diagnóstico (meses) 12 60 108 63 4 Sexo masculino Sim Sim Sim Sim Sim Dias de febre 7 6 8 9 14 DK clássico Sim Sim Sim Sim Não Dia de febre na 1ᵃ dose IGIV 7 6 4 6 14 Resistência ao tratamento IGIV Não Não Sim Sim NA Dia de febre na 2ᵃ dose IGIV NA NA 6 8 NA MPDN (30mg/kg/dia) Com 1ᵃ dose IGIV Com 2ᵃ dose IGIV Não NA NA Não NA NA Não Não Sim Sim Não Sim Sim Sim Não Classificação AAC Pequenos Pequenos Pequenos Médios Gigantes Z escore máximo NA 4,46 3,56 6,94 13,81 Artérias atingidas ACD; TC ACD; ACE ACE ACE; ACD ACD; CIR; DAE AAC: aneurismas das artérias coronárias; ACD: artéria coronária direita; ACE: artéria coronária esquerda; CIR: artéria circunflexa; DAE: descendente anterior esquerda; DK: doença de Kawasaki; IGIV: imunoglobulina intravenosa; MPDN: metilprednisolona; NA: não aplicável; TC: tronco comum. Na fase aguda, para além do envolvimento coronário, observou-se derrame pericárdico em 10 casos, insuficiência valvular mitral ligeira em três e disfunção ventricular em três, um dos quais com choque cardiogênico. Um apresentou bloqueio atrioventricular (BAV) de 1ºgrau variável. Após a fase aguda, um paciente manteve envolvimento do sistema de condução e dilatação do ventrículo esquerdo (VE) e um ficou com hipertrofia do VE. Os valores de S, E, valor preditivo positivo (VPP) e valor preditivo negativo (VPN) para os diferentes modelos estão apresentados na tabela 5, sendo apenas incluídos os pacientes a quem foi possível categorizar com alto ou baixo risco. Tabela 5 Sensibilidade, especificidade, valores preditivos positivos e negativos dos diferentes modelos Modelo n S E VPP VPN Kobayasahi 34 63,6% 77,3% 53,8% 81% Egami 39 66,7% 73,1% 50% 82,6% Sano 25 28,6% 94,1% 66,7% 77,3% E: especificidade; n: número de casos incluídos; S: sensibilidade; VPN: valor preditivo negativo; VPP: valor preditivo positivo. Discussão A DK é uma vasculite que, embora não tendo uma incidência tão elevada como no Japão, é, ainda assim uma causa importante de doença em idade pediátrica na nossa população. Um diagnóstico e introdução de terapêutica precoces constituem dois fatores muito importantes para reduzir o risco de envolvimento cardíaco. O presente estudo revelou uma percentagem de casos resistentes à IGIV coincidentes com os 10 a 20% descritos na literatura.2 Ao longo dos anos têm sido desenvolvidos esforços no sentido de encontrar fatores clínicos e laboratoriais que possam prever esta resistência de modo a introduzir mais precocemente terapêuticas coadjuvantes. Existem diversos parâmetros descritos na literatura tais como idade, albumina, transaminases, bilirrubina total, neutrófilos, plaquetas, PC-R, VS, entre outros.14,16–20 Neste estudo observou-se que a PC-R e a VS apresentaram capacidade preditora estatisticamente significativa de resistência à IGIV. No caso da PC-R, o ponto de corte ótimo foi de 15,1 mg/dL, com uma sensibilidade de 0,778, especificidade de 0,789 e um OR de 13,125. Pacientes com PC-R superior a 15,1mg/dL apresentam uma probabilidade de resistência à IGIV cerca de 13 vezes superior aos que têm valores inferiores. Relativamente à VS, o ponto de corte ótimo foi de 90,5 mm/h, com uma sensibilidade de 0,667, especificidade 0,857 e OR 12,000. Pacientes com VS superior a 90,5 mm/h apresentam uma probabilidade de resistência à IGIV cerca de 12 vezes superior aos que têm valores inferiores. Combinando estas duas variáveis independentes obteve-se um modelo estatisticamente significativo (p = 0,042), cujo ponto de corte apresenta uma sensibilidade de 0,833 e especificidade de 0,771. No entanto, a variância explicada pelo modelo é apenas de 25% (Nagelkerke R2 = 0,254), pelo que, embora seja estatisticamente significativo, não pode ser validado, o que se deve em grande parte ao tamanho reduzido da amostra. Todavia, é importante realçar esta tendência no sentido de que as duas variáveis poderão ser importantes para prever a resistência à IGIV, antes da sua infusão. O fato destas variáveis terem sido preditoras de resistência, realça o papel da inflamação exuberante nesta doença, etiologia que é defendida como possível precipitante da resposta imunológica que culmina numa vasculite.21 A etiologia da DK continua incerta, no entanto vários fatores são apontados como predisponentes. Um deles é a imaturidade do sistema imunitário, condição que é apoiada pelo fato de afetar predominantemente crianças com idade inferior a cinco anos. Neste estudo 62,5% dos pacientes pertenciam a esta faixa etária, o que, embora corresponda à maioria da amostra, fica aquém dos 80% descritos na literatura.1 Uma possível explicação para estes resultados é a contribuição genética, sendo as percentagens reportadas baseadas em estudos com variedade étnica alargada, incluindo asiáticos. Verificou-se envolvimento coronário em 25% dos casos, com sete a cumprir critérios de dilatação e cinco de aneurismas das artérias coronárias. Os 10% de incidência de AAC, foram superiores aos 4% reportados para os casos devidamente tratados. Comparando os grupos com e sem envolvimento coronário, verificou-se diferença estatisticamente significativa relativamente ao tempo total de febre (p = 0,038). Este resultado corrobora a ideia da persistência da febre ser deletéria e a necessidade da administração de IGIV preferencialmente até ao 10º dia para evitar sequelas cardíacas.9 O uso de corticosteróide na DK continua a ser tema de debate e controvérsia. O mais consensual é usar metilprednisolona por via intravenosa (MPDN iv) na dose de 15 a 30mg/kg/dia durante três dias.9 A MPDN iv, nos pacientes com DK refratária à IGIV, suprime os níveis de citocinas inflamatórias mais rapidamente que uma 2ᵃ dose de IGIV,9 no entanto não está recomendada como primeira linha. Sleeper et al.10 avaliaram o uso de corticosteróide em diferentes momentos, observando-se diferença estatisticamente significativa no aparecimento de AAC apenas nos pacientes refratários à IGIV e que levaram a administração de segunda dose. Neste estudo também se avaliaram complicações cardíacas e achados ecográficos que não o envolvimento coronário. Das complicações cardíacas em fase aguda mais graves destacaram-se três casos de disfunção ventricular esquerda, um com choque cardiogénico e um BAV de 1º grau, complicações essas também descritas na literatura.2,7 Na fase aguda, foi ainda possível observar 10 pacientes com derrame pericárdico sem compromisso hemodinâmico e três com insuficiência valvular mitral ligeira. Em Paris, Chbeir et al.22 obtiveram relação estatisticamente significativa entre resistência à IGIV, AAC e achados na ecografia cardíaca inicial, tais como derrame pericárdico, hiperecogenicidade das coronárias e dilatação coronária. Os autores desse estudo não consideraram a hiperecogenicidade e o afunilamento das coronárias como fatores relevantes, visto que são achados subjetivos, pouco reprodutíveis e podem ser encontrados tanto em doenças febris como em crianças saudáveis.23 Na fase crônica, um paciente manteve envolvimento do sistema de condução e dilatação do VE e outro ficou com hipertrofia do VE. As repercussões cardíacas a longo prazo na DK estão ainda pouco esclarecidas. Friedman et al.6 mostraram maior ocorrência de efeitos adversos cardíacos a longo prazo, tais como morte, transplante cardíaco, cirurgias de bypass coronário e angioplastia primária em pacientes com DK que desenvolveram AAC com escores z superiores e que foram resistentes à IGIV. Um estudo realizado por Holve et al.8 revelou baixa incidência de efeitos adversos cardíacos até os 21 anos de idade, mas uma maior probabilidade de desenvolver hipertensão arterial após os 15 anos de idade. Os modelos validados no Japão apresentaram fraca utilidade clínica no presente estudo (Tabela 4). O modelo com maior especificidade foi o de Sano, embora com sensibilidade muito baixa e apenas com um pequeno número de casos incluído. O modelo de Egami foi o mais sensível para esta amostra, mas, ainda assim, insuficiente para ser validado. Na base dessa diferença de resultados, pode estar o componente genético. Aliás esses resultados vão ao encontro de outros trabalhos realizados fora do Japão, em que nenhum deles conseguiu validar os modelos nas suas amostras.10,14–17,20 É necessário levar em consideração ainda que existiram algumas diferenças no desenho do estudo relativamente aos modelos japoneses, nomeadamente aplicados apenas a pacientes com DK clássica. Um estudo japonês falhou em validar os modelos em uma amostra de pacientes apenas com DK atípica.24 O modelo de Kobayashi foi validado na população japonesa com uma sensibilidade de 0,86 e especificidade de 0,67.11 Ao contrário do presente estudo, a IGIV foi administrada na dose de 1 g/kg em dois dias consecutivos e foi considerada resistência se febre persistente após 24 horas de início de terapêutica, ou se recrudescência com sintomas após período apirético. O modelo de Egami foi validado com uma sensibilidade de 0,78 e especificidade de 0,76; no entanto, foi definida resistência se ausência de diminuição do valor de PC-R em mais de 50% e persistência da febre 48 horas após administração de IGIV.12 Loomba et al.25 não conseguiram validar o modelo de Egami, mesmo aplicando-o separadamente à DK clássica, atípica e por etnias. O modelo de Sano, validado com uma sensibilidade de 0,77 e especificidade de 0,86, foi o único dos três a ajustar o tamanho dos AAC à superfície corporal.13 No entanto, também usou administração de IGIV na dose de 1 g/kg em 2 dias consecutivos e definiu resistência se persistência de febre após 24 horas do término da terapêutica. Este trabalho apresenta as limitações inerentes ao fato de ser um estudo retrospectivo e com uma amostra de tamanho reduzido. Conclusão A PC-R e a VS são variáveis independentes que mostraram tendência preditora de resistência à IGIV com pontos de corte ótimos de 15,1 mg/dL e 90,5 mm/h, respectivamente. Existe, no entanto, necessidade de um estudo com uma amostra de dimensões adequadas para validar um modelo baseado nesses dois dados analíticos. As complicações cardíacas não se resumem às artérias coronárias, devendo ser mais abrangentes o estudo e o seguimento desses pacientes. Os modelos validados para a população japonesa apresentam utilidade muito limitada na amostra deste estudo, reforçando ainda mais a necessidade e a importância de novas abordagens. Original Article Kawasaki Disease: Predictors of Resistance to Intravenous Immunoglobulin and Cardiac Complications https://orcid.org/0000-0001-7047-4170 Faim Diogo 1 https://orcid.org/0000-0001-7506-0647 Henriques Cláudio 1 Brett Ana 2 Francisco Andreia 1 Rodrigues Fernanda 2 Pires António 1 1 Coimbra Portugal Centro Hospitalar e Universitário de Coimbra EPE - Cardiologia Pediátrica Coimbra - Portugal 2 Coimbra Portugal Centro Hospitalar e Universitário de Coimbra EPE - Urgência e Unidade de Infeciologia, Coimbra - Portugal Mailing Address: Diogo Faim • Centro Hospitalar e Universitário de Coimbra EPE - Praceta, R. Prof. Mota Pinto, 3075 Coimbra 3000-075 – Portugal. E-mail: diogofaim92@gmail.com Abstract Background: Kawasaki disease (KD) is the leading cause of acquired cardiac disease in children, in developed countries. Objectives: To identify predictive factors for resistance to intravenous immunoglobulin (IVIG), calculate the effectiveness of Japanese predictive models and characterize cardiac complications. Methods: Retrospective analysis of KD cases admitted in a Portuguese paediatric hospital between january 2006 and july 2018. ROC curves were used to determine predictive factors for resistance and the multivariate logistic regression analysis was used to develop the predictive model. A significance level of 5% was used. Results: 48 patients with a median age of 36 months were included. The IVIG resistance was 21%. Echocardiographic anomalies were noted in 46%, with coronary involvement in 25% of the sample population. As predictive variable of resistance, the C-reactive protein (CRP) presented an AUC ROC = 0.789, optimal cut-off value 15.1 mg/dL, sensitivity (Sn) 77.8% and specificity (Sp) 78.9%. The erythrocyte sedimentation rate (ESR) presented an AUC ROC = 0.781, optimal cut-off value 90.5 mm/h, Sn 66.7% and Sp 85.7%. The model with the two variables showed p = 0.042 and AUC ROC = 0.790. Predictive strength of Japanese models were: Kobayashi (Sn 63.6%, Sp 77.3%), Egami (Sn 66.7%, Sp 73.1%), Sano (Sn 28.6%, Sp 94.1%). Conclusion: CRP and ESR are independent variables that were related to IVIG resistance, with optimal cut-off points of 15.1 mg/dL and 90.5 mm/h, respectively. About half of the patients had some form of cardiac involvement. The Japanese models appeared to be inadequate in our population. (Arq Bras Cardiol. 2021; 116(3):485-491) Keywords: Kawasaki Disease/complications Mucocutaneous Lymph Node Syndrome/complications Drug Resistance Coronary Artery Disease Immunoglobulin Child Introduction Kawasaki disease (KD) is an acute self-limiting vasculitis, which affects medium-sized vessels and is the leading cause of acquired cardiac disease in pediatric age groups.1 Its etiology remains uncertain, but several factors have been associated to it, namely genetic, environmental, and inflammatory ones.2 Although with a worldwide distribution, its highest prevalence is in Japan, where the incidence is on the rise.3 In Portugal, an epidemiological study carried out in 2017 showed an mean annual incidence of 6.5 per 100,000 children under 5 years of age.4 Based on the 2004 American Pediatric Academy criteria,5 classic KD is considered if fever persists for five days or more and if at least four of five additional clinical criteria are observed: nonexudative bilateral conjunctivitis, alterations of the lips and oral cavity, erythematous rash, changes in the extremities, and cervical lymphadenopathy. If fever lasts for five or more days and only two or three additional criteria are present, it is considered atypical KD, if supported by laboratory and echocardiographic data.2 If not treated within an established period, KD can be complicated by coronary artery aneurysms (CAA) in up to 25% of cases.2 Although coronary artery involvement is the most feared consequence of the disease, other cardiac complications are possible.2,6–8 Treatment with intravenous immunoglobulin (IVIG) in the acute phase administered in the first 10 days of illness reduces the incidence of CAA to 4%.2 IVIG resistance occurs in 10-20% of cases, increasing the likelihood of coronary involvement.2 There are different possible approaches in case of IVIG resistance, such as a second dose of IVIG, corticosteroids and/or monoclonal antibodies.9 No benefit has been described when corticosteroids are used in addition to IVIG in the first instance and this therapeutics is currently reserved for refractory cases.10 In order to identify the cases that could potentially be resistant to treatment with IVIG, and benefit from adjuvant therapies in the initial phase, models based on a scoring system have been developed. Three have been validated in the Japanese population, namely the Kobayashi,11 Egami,12 and Sano scoring systems.13 However, several studies have shown that these models are poor predictors in many western populations.10,14,15 The aim of this study was to identify clinical and laboratory predictive factors regarding resistance to IVIG and coronary artery involvement, and to develop a more suitable predictor model of resistance in this population. Secondary objectives regard characterizing the KD cases admitted to a central pediatric hospital over a period of 13 years, to verify the effectiveness of the Japanese scoring systems in our population sample and to analyze the non-coronary cardiac complications of KD. Methods Sample Retrospective analysis of KD cases admitted to the Hospital Pediátrico – Centro Hospitalar e Universitário de Coimbra (HP-CHUC) diagnosed from 01/01/2006 to 06/30/2018. All patients between 30 days and <18 years with KD and treated with IVIG at diagnosis at HP-CHUC were included in the study. All the patients transferred from outlying centers with a diagnosis of KD and managed at these institutions were excluded. The diagnosis of typical and atypical KD was based on the American Academy of Pediatrics criteria. We considered day one of fever on the day the fever started, defined as axillar temperature ≥ 38ºC. Resistance to IVIG was considered if the fever persisted 36 hours after its administration. All patients who received corticosteroids simultaneously with the first dose of IVIG were excluded from the resistance quantification. Dallaire z scores were used to classify the coronary artery morphology defining: coronary artery ectasia if z score between 2 and 2.4, small aneurysm if z score between 2.5 and 4.9, medium aneurysm if z score between 5 and 9.9 and absolute dimension < 8 mm, and giant aneurysm if z score ≥ 10 or absolute dimension ≥ 8 mm. If unable to calculate the z score, the absolute dimensions were used, being small aneurysm if ≥ 2.5 mm and < 4mm, medium aneurysm if ≥ 4 mm and < 8 mm and giant aneurysm if ≥ 8 mm. Regarding other cardiac complications, the coronary artery hyperechogenicity and lack of tapering on echocardiography were not considered as echocardiographic diagnostic criteria. To calculate the effectiveness of the Japanese models, all patients who did not have the necessary data to be considered as high or low risk of IVIG resistance were excluded. Scoring and categorization in high or low risk patients were performed as shown in Table 1. Table 1 Scoring system of the Japanese modelss Model Score High risk Kobayashi ≥ 4 points AST > 100 U/L 2 Na ≤ 133 mmol/L 2 IVIG with fever ≤ 4 days 2 Neutrophils/Leucocytes ≥ 80% 2 CRP ≥ 10 mg/dL 1 Age ≤ 1 year old 1 Platelets ≤ 300,000/µL 1 Egami ≥ 3 points ALT ≥ 80 U/L 2 IVIG with fever ≤ 4 days 1 CRP ≥ 8 mg/dL 1 Age ≤ 6 months 1 Platelets ≤ 300,000/µL 1 Sano ≥ 2 points AST > 200 U/L 1 Total bilirubin ≥ 0.9 mg/dL 1 CRP ≥ 7 mg/dL 1 AST: aspartate transaminase; U: international unity; L: liter; Na: serum sodium; mmol: millimole; IVIG: intravenous immunoglobulin; CRP: C-reactive protein; mg: milligram; dL: deciliter; µL: microliter; ALT: alanine transaminase. Statistical Analysis The SPSS® (IBM®, SPSS® Statistics Inc., Chicago) program version 25.0 was used the performed the statistical analysis. The Shapiro-Wilk test was used to test the normality of the variables. The continuous variables with normal distribution were described using mean and standard deviation (SD) and continuous variables without normal distribution were described using median and interquartile range (IQR). We used the Fisher’s exact test to compare categorical variables, the Student’s t-test to compare parametric variables and the Mann-Whitney test to compare the non-parametric ones. The Receiver Operating Characteristic (ROC) curves were used to evaluate the individual discriminative capacity of each variable and to identify the optimal cutoff points to predict resistance to IVIG. The variables were considered as good predictors if the area under the curve (AUC) > 0.75. Multivariate logistic regression analysis was used to develop the predictive resistance model. A significance level of 5% was used. Results Forty-eight patients met the KD criteria, of whom 32 (66.7%) were male. The median age was 36 months (IQR 16.75-89.25), 62.5% of patients were less than five years old and 10.4% over nine years old. On the day of admission, all the patients presented with fever, with a median duration of five days (IQR 4-8), minimum of one day and maximum of 14 days. Among the five main clinical criteria, nonexudative bilateral conjunctivitis was observed in 94% of cases, alterations of the lips and oral cavity in 90%, erythematous rash in 84%, changes in extremities in 75%, and cervical lymphadenopathy in 69%. The most common findings among oral alterations were cheilitis (67%) and lip erythema (67%), followed by erythema of the oropharynx (50%) and the strawberry tongue (48%). The most prevalent changes in the extremities were erythema (52%), followed by swelling (31%) and peeling (25%). Inflammatory signs at the Bacillus Calmette-Guérin (BCG) vaccination site were observed in 23% of patients. Atypical KD was diagnosed in 17% of the cases. The median duration of hospital stay was two days (IQR 1-6.75). During the acute phase, IVIG 2g/kg was administered to all patients. The median day of illness of the administration was 6.5 (IQR 5-8). Simultaneously, 45-100mg/kg of acetylsalicylic acid (ASA) were administered to 47 patients in the acute phase. Five children received corticosteroids together with the first dose of IVIG. After the acute phase, all patients were medicated with ASA 3-5 mg/kg/day, three patients with clopidogrel and one with enoxaparin. Nine patients were resistant to the IVIG (21%), of which one had atypical KD (p = 0.543). All nine repeated IVIG administration, five of which with methylprednisolone 30 mg/kg/day. Among the variables evaluated as predictors of IVIG resistance (Table 2), C-reactive protein (CRP) presented an AUC ROC of 0.78 (95% confidence interval (CI): 0.632 – 0.947), and the erythrocyte sedimentation rate (ESR), an AUC ROC of 0.781 (95%CI: 0.585 – 0.977). The optimal cut-off value for CRP was 15.1 mg/dL with sensitivity (Sn) of 77.8% and specificity (Sp) of 78.9% (Odds ratio (OR) = 13.125 95%CI: 2.271 – 75.858). The optimal cut-off value for ESR was 90.5 mm/h, with Sn of 66.7% and Sp of 85.7% (OR = 12.000 95%CI: 1.718 – 83.803). A logistic model was developed with these two variables, with a p-value of 0.042, AUC ROC of 0.79 (95%CI: 0.589 – 0.992), with Sn of 83.3% and Sp of 77.1%, but with a 25% variance (Nagelkerke R2 = 0.254). Table 2 Receiver operating characteristic analysis of several variables to predict resistance to intravenous hemoglobulin Characteristic AUC [95%CI] Age 0.542 [0.377; 0.708] IVIG administration day 0.595 [0.403; 0.787] Hemoglobin 0.611 [0.416; 0.806] Leucocytes 0.525 [0.331; 0.719] Neutrophils 0.637 [0.447; 0.828] Platelets 0.513 [0.295; 0.732] ESR 0.781 [0.585; 0.977] CRP 0.789 [0.632; 0.947] Na 0.715 [0.475; 0.955] AST 0.648 [0.434; 0.862] ALT 0.693 [0.486; 0.901] Total bilirubin 0.500 [0,139; 0.861] Albumin 0.693 [0.459; 0.928] AUC: area under the curve; CI: confidence interval; IVIG: intravenous immunoglobulin; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; Na: serum sodium; AST: aspartate transaminase; ALT: alanine transaminase. Coronary artery changes were found in 12 children (25%), seven with ectasia and five with CAA. The comparison between groups with and without coronary artery involvement is shown in Table 3. The duration of fever and the use of corticosteroids were the only significant differences between these two groups. Patients with coronary artery involvement had longer duration of fever (p = 0.038) and greater need for corticotherapy (p = 0.009). Four patients had CAA when methylprednisolone was started. Among the five patients with CAA, three met the criteria for small aneurysms, one for medium aneurysms and one for giant ones. These patients are summarized in Table 4. Table 3 Characteristics of groups with and without coronary artery involvement Characteristics Without coronary involvement (n =36) Coronary involvement (n= 12) p - value Corticoid (n=10) n 4 6 0.009 Resistance to IVIG (n=9) n 5 4 0.173 Age (months) Mean ± sd 59.7 ± 57 47.5 ± 30.1 0.35 Fever duration (days) Mean ± sd 7.4 ± 2.8 9.4 ± 3 0.038 IVIG administration day Mean ± sd 6.5 ± 2.9 7.6 ± 3.5 0.283 Atypical KD n 6 2 0.686 Haemoglobin (g/dL) Mean ± sd 11.5 ± 1.3 11.2 ± 1.3 0.359 Leucocytes (/µL) Mean ± sd 14,174 ± 6,010 15,216 ± 6,918 0.619 Neutrophils (/µL) Mean ± sd 9,515 ± 4,770 10,994 ± 5,629 0.378 Platelets (/µL) Mean ± sd 328,389 ± 127,125 362,667 ± 282,652 0.691 CRP (mg/dL) Mean ± sd 10.4 ± 8.8 19.6 ±25 0.243 ESR (mm/h) Mean ± sd 71.6 ± 19 76.7 ± 33.4 0.672 Na (mmol/L) Mean ± sd 137 ± 4 137 ± 5 0.869 AST (U/L) Mean ± sd 64 ± 53 101 ± 91 0.222 ALT (U/L) Mean ± sd 99 ± 116 113 ± 86 0.712 Total bilirubin (mg/dL) Mean ± sd 1.9 ± 2 2.8 ± 3 0.538 Albumin (g/L) Mean ± sd 35.6 ± 4.3 34.9 ± 7.2 0.77 n: absolute value; IVIG: intravenous immunoglobulin; sd: standard deviation; KD: Kawasaki disease; g: gram; dL: deciliter; µL: microliter; CRP: C-reactive protein; mg: milligram; ESR: erythrocyte sedimentation rate; h: hour; Na: serum sodium; mmol: millimole; L: litre; AST: aspartate transaminase; U: international unity; ALT: alanine transaminase. Table 4 Characteristics of patients with coronary artery aneurysms Characteristics Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Age (months) 12 60 108 63 4 Male Yes Yes Yes Yes Yes Fever duration (days) 7 6 8 9 14 Classic KD Yes Yes Yes Yes No Day of fever in IVIG 1st dose 7 6 4 6 14 Resistance to IVIG No No Yes Yes NA Day of fever in IVIG 2nd dose NA NA 6 8 NA MPDN (30mg/Kg/day) With IVIG 1st dose With IVIG 2nd dose No NA NA No NA NA Yes No Yes Yes No Yes Yes Yes No CAA classification Small Small Small Medium Giant Maximum z score NA 4.46 3.56 6.94 13.81 Arteries involved RCA; CT RCA; LCA LCA RCA; LCA RCA; LAD; LCX KD: Kawasaki disease; IVIG: intravenous immunoglobulin; MPDN: methylprednisolone; mg: milligram; Kg: kilogram; CAA: coronary artery aneurysms; RCA: right coronary artery, CT: common trunk; LCA: left coronary artery; LAD: left anterior descending artery; NA: not applicable; LCX: left circumflex artery. In the acute phase, in addition to coronary involvement, 10 patients presented with pericardial effusion, three with mild mitral valve regurgitation, two with left ventricular systolic dysfunction, one with cardiogenic shock and one with variable first degree atrioventricular (AV) block. After the acute phase, the patient with the AV block developed left ventricle (LV) dilation and another patient developed LV hypertrophy. Table 5 summarizes Sn, Sp,and the positive (PPV) and negative predictive values (NPV) for the Japanese models in our sample. Table 5 Statistical values of the Japanese models in our study Model n Sn (%) Sp (%) PPV (%) NPV (%) Kobayasahi 34 63.6 77.3 53.8 81 Egami 39 66.7 73.1 50 82.6 Sano 25 28.6 94.1 66.7 77.3 n: absolute value; Sn: sensitivity; Sp: Specificity; PPV: positive predictive value; NPV: negative predictive value. Discussion Despite the lower incidence compared to Japan, KD is a vasculitis that is still an important cause of pediatric disease in our population. Early diagnosis and management are two important factors that appear to reduce cardiac involvement. Our study revealed an incidence of IVIG resistance similar to the 10 to 20% described in the literature.2 Over the years, efforts have been made to find clinical and laboratory factors that can predict this resistance in order to introduce adjuvant therapies at an early stage of the disease. There are several parameters in the literature that have been studied for this purpose, such as age, serum albumin, transaminases, total bilirubin, neutrophils count, platelet count, CRP, ESR, among others.14,16–20 In our study, CRP and ESR presented a statically significant predictive capacity in relation to IVIG resistance. For CRP, the optimal cut-off point was 15.1 mg/dL (Sn 77.8%, Sp 78.9%, OR 13.125). Patients with CRP values above 15.1 mg/dL are about 13 times more likely to be resistant to IVIG than those with lower values. Concerning ESR, the optimal cut-off point was 90.5 mm/h (Sn 66.7%, Sp 85.7%, OR 12.000). Patients with ESR greater than 90.5 mm/h have a probability of resistance to IVIG approximately 12 times higher than those with lower values. Combining these two independent variables, a statistically significant model was obtained (p = 0.042), whose cut-off point has Sn of 83.3% and Sp of 77.1%. Despite these encouraging results, the variance explained by the model is only 25% (Nagelkerke R2 = 0.254). Thus, although statistically significant, it cannot be validated, which is largely due to the small sample size. Nevertheless, based on these trends, the base-line values for CRP and ESR should be known prior to IVIG administration. The resistance predictor capacity highlights the role of inflammation in this disease, a possible underlying trigger in KD vasculitis.21 The etiology of KD remains uncertain, however, predisposing factors have been put forward. One, is the immaturity of the immune system, a theory that is supported by the fact that KD predominantly affects children under the age of five. In our study, 62.5% of the patients belonged to this age group, which, although corresponding to the majority of the sample, is below the 80% described in the literature.1 A possible explanation for this result is genetic contribution, since the incidences described in the literature are from studies with a wide range of ethnicities, including Asian children. Coronary artery involvement occurred in 12 (25%) children, seven with ectasia and five with CAA. Therefore, the incidence of CAA was 10%, which is higher than the 4% reported in the literature. Comparing the groups with and without coronary artery involvement, a statistically significant difference was found regarding the duration of fever (p = 0.038). This result highlights the deleterious effects of persistent fever and the need for IVIG administration, preferably up to the tenth day of the disease, in order to avoid cardiac sequelae.9 The use of corticosteroids in KD is still a topic of debate and controversy. The most consensual is the use intravenous methylprednisolone (MPDN) at a dose of 15 to 30 mg/kg/day, for three days.9 In patients with refractory KD, MPDN supresses the inflammatory cytokine levels more quickly than a second dose of IVIG,9 although it is not recommended as a first-line treatment. Sleeper et al.10 evaluated the impact of corticosteroids at different times of the disease and showed that, with regards to the development of CAA, the only statistically significant difference was in those refractory to the first dose of IVIG which combined corticosteroids with the second dose of IVIG. The cardiac complications and echocardiographic findings, others than coronary artery involvement were also evaluated. Three cases of left ventricular systolic dysfunction were identified, one of which with cardiogenic shock, a complication also described in the literature.2,7 In the acute phase, ten patients presented with pericardial effusion without hemodynamic compromise, three patients with mild mitral valve regurgitation and one with first degree AV block. Chbeir et al.22 found a relation between resistance to IVIG, CAA, and initial cardiac echocardiographic findings such as pericardial effusion, coronary hyperechogenicity, and coronary ectasia. The coronary hyperechogenicity and the lack of tapering on echocardiography were not considered as relevant factors, since they are subjective findings, poorly reproducible and can be found both in febrile illnesses and in healthy children.23 During the chronic phase, one patient remained with conduction system impairment and developed LV dilation, and another patient developed LV hypertrophy. The long-term cardiac repercussions in KD remain unclear. Friedman et al.6 reported an increase in the occurrence of long-term adverse cardiac effects, leading to primary angioplasty, coronary bypass surgery, heart transplantation, and death, in patients who developed CAA with higher z scores and who were initially resistant to IVIG. A study by Holve et al.8 revealed a low incidence of adverse cardiac effects in subjects up to 21 years of age, but a greater likelihood of developing high blood pressure from the age of 15. Japanese predictive models presented poor clinical utility in this study (Table 4). The model with the highest specificity was Sano’s, although with very low sensitivity and with only a small number of cases included. Egami’s model was the most sensitive, however, not powerful enough to be validated. The genetic component may be the explanation for these differences. In fact, the results presented here are similar to those of other studies carried out outside Japan, in which none were able to validate the models in their samples.10,14–17,20 It is still important to note the differences in the study design in relation to the Japanese models, namely that they were applied only to patients with classical KD. Another Japanese study failed to validate the models in a sample exclusively composed of atypical KD cases.24 The Kobayashi model was validated for the Japanese population with Sn of 86% and Sp of 67%.11 Contrary to the present study, IVIG was administered at a dose of 1 g/kg on two consecutive days and resistance was considered if fever persisted 24 hours after the beginning of the treatment, or in case of recurrence after a period without fever. The Egami model was validated with Sn of 78% and Sp of 76%, however, resistance was considered if the CRP value did not decrease by more than 50% and fever persisted for longer than 48 hours after IVIG administration.12 Loomba et al.25 were not able to validate the Egami model even when applying it separately to classical and atypical KD, and by ethnicity. The Sano model, validated with Sn of 77% and Sp of 86%, was the only one of the three to adjust the size of the CAA to the body surface.13 However, it also used IVIG at a dose of 1 g/kg on two consecutive days and defined resistance if fever persisted 24 hours after the end of therapy. The main limitations of this analysis are related to its retrospective methodology and sample size. Conclusions CRP and ESR are independent variables that showed a predictive trend regarding resistance to IVIG, with optimal cut-off values of 15.1 mg/dL and 90.5 mm/h, respectively. However, there is a need for a multicenter study with a sample of adequate dimensions to validate a model based on these two analytical parameters. Cardiac complications are not limited to coronary arteries, and the study and follow-up of these patients should be more widespread. The validated models for the Japanese population have very limited utility in our population, further reinforcing the need and importance of new approaches. Fontes de Financiamento O presente estudo não contou com fontes de financiamento externas. Vinculação Acadêmica Não há vinculação deste estudo a programas de pós-graduação. Aprovação Ética e Consentimento Informado Este artigo não contém estudos com humanos ou animais realizados por nenhum dos autores. Author Contributions Conception and design of the research: Faim D, Henriques C, Brett A, Francisco A, Rodrigues F, Pires A; Data acquisition: Faim D, Henriques C; Analysis and interpretation of the data: Faim D, Brett A, Francisco A; Statistical analysis and Writing of the manuscript: Faim D; Critical revision of the manuscript for intellectual content: Faim D, Brett A, Francisco A, Rodrigues F, Pires A. Potential Conflict of Interest The authors report no conflict of interest concerning the materials and methods used in this study or the findings specified in this paper. Sources of Funding There was no external funding source for this study. Study Association This study is not associated with any thesis or dissertation. Ethics Approval and Consent to Participate This article does not contain any studies with human participants or animals performed by any of the authors.	3?5MILLIGRAM/KILOGRAM/DAY	DrugDosageText	CC BY	33470332	19,013,675	2021-03
What was the dosage of drug 'HUMAN IMMUNOGLOBULIN G'?	Kawasaki Disease: Predictors of Resistance to Intravenous Immunoglobulin and Cardiac Complications. Kawasaki disease (KD) is the leading cause of acquired cardiac disease in children, in developed countries. To identify predictive factors for resistance to intravenous immunoglobulin (IVIG), calculate the effectiveness of Japanese predictive models and characterize cardiac complications. Retrospective analysis of KD cases admitted in a Portuguese paediatric hospital between january 2006 and july 2018. ROC curves were used to determine predictive factors for resistance and the multivariate logistic regression analysis was used to develop the predictive model. A significance level of 5% was used. 48 patients with a median age of 36 months were included. The IVIG resistance was 21%. Echocardiographic anomalies were noted in 46%, with coronary involvement in 25% of the sample population. As predictive variable of resistance, the C-reactive protein (CRP) presented an AUC ROC = 0.789, optimal cut-off value 15.1 mg/dL, sensitivity (Sn) 77.8% and specificity (Sp) 78.9%. The erythrocyte sedimentation rate (ESR) presented an AUC ROC = 0.781, optimal cut-off value 90.5 mm/h, Sn 66.7% and Sp 85.7%. The model with the two variables showed p = 0.042 and AUC ROC = 0.790. Predictive strength of Japanese models were: Kobayashi (Sn 63.6%, Sp 77.3%), Egami (Sn 66.7%, Sp 73.1%), Sano (Sn 28.6%, Sp 94.1%). CRP and ESR are independent variables that were related to IVIG resistance, with optimal cut-off points of 15.1 mg/dL and 90.5 mm/h, respectively. About half of the patients had some form of cardiac involvement. The Japanese models appeared to be inadequate in our population. (Arq Bras Cardiol. 2021; 116(3):485-491). Introdução A doença de Kawasaki (DK) é uma vasculite aguda e autolimitada que afeta os vasos de média dimensão, sendo a principal causa de cardiopatia adquirida em idade pediátrica.1 A sua etiologia continua incerta, mas vários fatores têm sido associados à doença, nomeadamente genéticos, ambientais e inflamatórios.2 Embora com distribuição mundial, a sua maior prevalência é no Japão, com incidência crescente.3 Em Portugal, um estudo epidemiológico realizado em 2017 descreveu uma incidência anual média de 6,5 por 100.000 crianças com menos de 5 anos de idade.4 Com base nos critérios da American Pediatric Academy de 2004,5 considera-se DK clássica se febre ≥ 5 dias e presença de pelo menos 4 de 5 critérios clínicos adicionais: conjuntivite bilateral não exsudativa, alterações dos lábios e da mucosa oral, exantema polimorfo, alterações das extremidades e linfadenopatia cervical não supurativa. Se febre ≥ 5 dias e apenas 2 ou 3 critérios adicionais, considera-se DK atípica e é necessário recorrer a dados laboratoriais e ecográficos para melhor sustentar o diagnóstico.2 Se não tratada atempadamente, a DK pode complicar-se com aneurismas das artérias coronárias (AAC) em até 25% dos casos.2 Apesar de o envolvimento coronário ser a consequência mais temida da doença, são possíveis outras complicações cardíacas.2,6–8 O tratamento com imunoglobulina intravenosa (IGIV) na fase aguda continua a ser a principal terapêutica e, se administrada nos primeiros 10 dias de doença, diminui a incidência de AAC para 4%.2 A resistência à IGIV ocorre em 10% a 20% dos casos, aumentado a probabilidade de envolvimento coronário.2 Há diferentes abordagens possíveis quando há resistência, nomeadamente uma segunda dose de IGIV, corticosteroides e/ou anticorpos monoclonais.9 Não há descrição de benefício na administração inicial de corticosteroide juntamente com a IGIV a todos os pacientes, sendo atualmente tal terapêutica reservada para os casos refratários.10 Com o objetivo de identificar os casos que potencialmente poderiam ser resistentes ao tratamento com IGIV, e como tal beneficiar de outras terapêuticas na fase inicial, foram desenvolvidos modelos com base em um sistema de escores, nomeadamente os de Kobayashi,11 Egami12 e Sano,13 que foram validados para a população japonesa. No entanto, diversos estudos mostraram que esses modelos são fracos preditores em diversas populações ocidentais.10,14,15 O objetivo desse estudo foi identificar fatores preditores clínicos e analíticos de resistência à IGIV e de envolvimento coronário e construir um modelo preditor de resistência mais adequado para essa população. Como objetivos adicionais, pretendeu-se caracterizar os casos de DK em um hospital pediátrico de nível III nos últimos anos, calcular a eficácia dos escores japoneses nessa amostra e analisar as complicações cardíacas não coronárias da DK. Métodos Amostra Estudo retrospectivo dos casos diagnosticados com DK desde 1º de janeiro de 2006 a 30 de junho de 2018 no Hospital Pediátrico – Centro Hospitalar e Universitário de Coimbra (HP-CHUC). Foram incluídas todas as crianças e adolescentes com idades entre 30 dias e < 18 anos, com diagnóstico e terapêutica de fase aguda realizados no HP-CHUC. Foram excluídos todos os pacientes transferidos para o HP-CHUC já com diagnóstico e/ou terapêutica realizados em outro hospital. Foram utilizados os critérios da American Academy of Pediatrics para diagnóstico de DK clássica e de DK atípica. Considerou-se D1 de febre o dia inaugural de febre, definida como temperatura axilar ≥ 38°C. Considerou-se resistência à IGIV se a febre persistiu 36 horas após a sua administração, tendo sido excluídos todos os casos em que foi administrado corticosteroide concomitantemente com a primeira dose de IGIV. Para classificar o envolvimento coronário, foram usados os escores z (desvios padrões) de Dallaire, definindo-se dilatação se escore z entre 2 e 2,4, aneurisma pequeno se escore z entre 2,5 e 4,9, aneurisma médio se entre 5 e 9,9 e dimensão absoluta inferior a 8 mm, aneurisma gigante se escore z ≥ 10 ou dimensão absoluta ≥ 8 mm. Nos casos em que a informação foi insuficiente para se calcular o escore z, utilizaram-se os valores absolutos, sendo aneurisma pequeno se ≥ 2,5mm e < 4mm, médio se ≥ 4mm e < 8mm e gigante se ≥ 8 mm. Nas complicações cardíacas, os achados ecocardiográficos de hiperecogenicidade e afunilamento das artérias coronárias não foram considerados. Para calcular a eficácia dos modelos, foram excluídos todos os pacientes que não apresentavam os dados necessários para serem considerados como alto ou baixo risco de resistência para determinado modelo. A pontuação e a categorização em pacientes de alto ou baixo risco foram realizadas conforme evidenciado na Tabela 1. Tabela 1 Sistema de pontuação dos modelos avaliados Escore Pontos Alto risco Kobayashi ≥ 4 pontos AST > 100 U/L 2 Na ≤ 133 mmol/L 2 IGIV com febre ≤ D4 2 Neutrófilos/Leucócitos ≥ 80% 2 PC-R ≥ 10 mg/dL 1 Idade ≤ 1 ano 1 Plaquetas ≤ 300.000/uL 1 Egami ≥ 3 pontos ALT ≥ 80 U/L 2 IGIV com febre ≤ D4 1 PC-R ≥ 8 mg/dL 1 Idade ≤ 6 meses 1 Plaquetas ≤ 300.000/uL 1 Sano ≥ 2 pontos AST > 200 U/L 1 Bilirrubina total ≥ 0,9 mg/dL 1 PC-R ≥ 7 mg/dL 1 ALT: alanina aminotransferase; AST: aspartato aminotransferase; dL: decilitro; L: litro; mg: miligrama; IGIV: imunoglobulina intravenosa; mmol: milimole; Na: sódio plasmático; PC-R: proteína C-reativa; U: unidade internacional; uL: microlitro. Análise Estatística Análise estatística efetuada com recurso ao programa SPSS® (IBM®, SPSS® Statistics Inc., Chicago), versão 25.0. Para testar a normalidade da amostra, foi utilizado o teste de Shapiro-Wilk. As variáveis contínuas com distribuição normal foram descritas por meio de média e desvio padrão, as variáveis contínuas sem distribuição normal foram descritas através de mediana e amplitude interquartil (AIQ). Para comparação de variáveis categóricas, foi utilizado o teste exato de Fisher; das variáveis numéricas paramétricas, o teste de t-student não pareado; e das variáveis numéricas não paramétricas, o teste de Mann-Whitney. Foram construídas curvas receiver operating characteristic (ROC) para avaliar a capacidade discriminativa individual de cada variável e identificar pontos de corte para a predição de resistência à IGIV. As variáveis foram consideradas preditoras se com uma area under the curve (AUC) superior a 0,75. Para a elaboração de um modelo preditor de resistência, foi utilizada a regressão logística multivariada. O nível de significância utilizado nesse estudo foi de 5%. Resultados Cumpriram critérios de DK 48 pacientes, sendo 32 (66,7%) do sexo masculino. A mediana de idades foi de 36 meses (AIQ 16,75-89,25), com 62,5% abaixo dos 5 anos de idade e 10,4% acima dos 9 anos. No dia de admissão, a totalidade dos pacientes apresentava-se com febre, com uma mediana de 5 dias de febre (AIQ 4-8), mínimo de 1 e máximo de 14 dias. Dos cinco critérios clínicos principais, observou-se conjuntivite não purulenta em 94%, alterações orais em 90%, exantema em 84%, alterações das extremidades em 75% e linfadenopatia cervical em 69%. Dentre as alterações orais, as mais prevalentes foram a queilite (67%) e o eritema labial (67%), seguidos de eritema da orofaringe (50%) e língua em framboesa (48%). Entre as alterações das extremidades, a mais prevalente foi eritema (52%), seguido de edema duro (31%) e descamação (25%). Os sinais inflamatórios no local de inoculação da vacina Bacillus Calmette-Guérin (BCG) foram descritos em 23%. Foi diagnosticada DK atípica em 17% dos casos. A mediana de dias de internamento foi de 2 dias (AIQ 1-6,75). A totalidade dos pacientes foi medicada em fase aguda com IGIV 2 g/kg com mediana do dia de administração de 6,5 dias (AIQ 5-8). Foram medicados em fase aguda 47 pacientes com ácido acetilsalicílico (AAS) em doses entre 45 a 100mg/kg/dia. Cinco pacientes foram ainda medicados com corticosteroide juntamente com a primeira dose de IGIV. Após a fase aguda, todos os pacientes foram medicados com AAS em dose de 3 a 5 mg/kg/dia, três pacientes com clopidogrel e um com enoxaparina. Verificou-se resistência à IGIV em nove casos (21%), dos quais havia um caso de DK atípica (p = 0,543). Dos nove pacientes resistentes à terapêutica, foi administrada uma segunda dose de IGIV 2 g/kg e a cinco destes, metilprednisolona na dose de 30 mg/kg/dia. Entre as diferentes variáveis avaliadas como preditoras de resistência (Tabela 2), a proteína C-reativa (PC-R) apresentou uma AUC ROC de 0,78 (IC95%: 0,632-0,947) e a velocidade de sedimentação (VS) uma AUC ROC de 0,781 (IC95%: 0,585-0,977). O ponto de corte para a PC-R foi de 15,1 mg/dL com uma sensibilidade (S) de 0,778 e especificidade (E) de 0,789 [Odds ratio (OR) = 13,125 IC95%: 2,271-75,858]. O ponto de corte para a VS foi de 90,5 mm/h, verificando-se uma sensibilidade de 0,667 e especificidade de 0,857 (OR = 12,000 IC95%: 1,718-83,803). Foi ajustado um modelo logístico com as duas variáveis PC-R e VS, modelo este que apresentou um valor p de 0,042 e AUC ROC de 0,790 (IC95%: 0,589-0,992). No ponto de corte ótimo, a sensibilidade foi de 0,833 e a especificidade foi de 0,771. Apresentou ainda uma variância de 25% (Nagelkerke R2 = 0,254). Tabela 2 Análise ROC de diversas variáveis para predizer resistência à IGIV Variável AUC [IC 95%] Idade 0,542 [0,377; 0,708] Dia de administração IGIV 0,595 [0,403; 0,787] Hemoglobina 0,611 [0,416; 0,806] Leucócitos 0,525 [0,331; 0,719] Neutrófilos 0,637 [0,447; 0,828] Plaquetas 0,513 [0,295; 0,732] VS 0,781 [0.585; 0,977] PC-R 0,789 [0,632; 0,947] Na 0,715 [0,475; 0,955] AST 0,648 [0,434; 0,862] ALT 0,693 [0,486; 0,901] Bilirrubina total 0,500 [0,139; 0,861] Albumina 0,693 [0,459; 0,928] ALT: alanina aminotransferase; AST: aspartato aminotransferase; AUC: área abaixo da curva; IC: intervalo de confiança; IGIV: imunoglobulina intravenosa; Na: sódio plasmático; PC-R: proteína C-reativa; ROC: receiver operating curve; VS: velocidade de sedimentação. Ocorreu envolvimento coronário em 12 casos (25%). Sete pacientes desenvolveram dilatação das artérias coronárias e cinco, AAC. Na Tabela 3, comparam-se os grupos sem e com envolvimento coronário. Observaram-se diferenças apenas na duração da febre e na utilização de corticosteroides. O tempo total de febre foi mais elevado no grupo com envolvimento coronário (p = 0,038). Esse grupo recebeu mais vezes corticosteroide (p = 0,009). Quatro desses pacientes já tinham diagnóstico de envolvimento coronário prévio à administração desse fármaco. Dos pacientes com AAC, três cumpriram critérios para aneurismas pequenos, um para aneurismas médios e um para aneurismas gigantes. Esses pacientes estão caracterizados na Tabela 4. Tabela 3 Características dos grupos com e sem envolvimento coronário Variáveis Sem envolvimento coronário (n =36) Envolvimento coronário (n = 12) p Corticosteroide (n = 10) N 4 6 0,009 Resistência à IGIV (n = 9) N 5 4 0,173 Idade (meses) Média ± DP 59,7 ± 57 47,5 ± 30,1 0,35 Dia de resolução da febre Média ± DP 7,4 ± 2,8 9,4 ± 3 0,038 Dia de administração de IGIV Média ± DP 6,5 ± 2,9 7,6 ± 3,5 0,283 DK atípica n 6 2 0,686 Hemoglobina (g/dL) Média ± DP 11,5 ± 1,3 11,2 ± 1,3 0,359 Leucócitos (/uL) Média ± DP 14.174 ± 6.010 15.216 ± 6.918 0,619 Neutrófilos (/uL) Média ± DP 9.515 ± 4.770 10.994 ± 5.629 0,378 Plaquetas (/uL) Média ± DP 32.8389 ± 12.7125 36.2667 ± 28.2652 0,691 PC-R (mg/dL) Média ± DP 10,4 ± 8,8 19,6 ±25 0,243 VS (mm/h) Média ± DP 71,6 ± 19 76,7 ± 33,4 0,672 Na (mmol/L) Média ± DP 137 ± 4 137 ± 5 0,869 AST (U/L) Média ± DP 64 ± 53 101 ± 91 0,222 ALT (U/L) Média ± DP 99 ± 116 113 ± 86 0,712 Bilirrubina total (mg/dL) Média ± DP 1,9 ± 2 2,8 ± 3 0,538 Albumina (g/L) Média ± DP 35,6 ± 4,3 34,9 ± 7,2 0,77 AIQ: amplitude interquartil; ALT: alanina aminotransferase; AST: aspartato aminotransferase; dL: decilitro; DK: doença de Kawasaki; DP: desvio padrão; g: grama; h: hora; IGIV: imunoglobulina intravenosa; L: litro; mg: miligrama; mmol: milimole; n: número absoluto; Na: sódio plasmático; p: valor p; PC-R: proteína C-reativa; U: unidade internacional; uL: microlitro; VS: velocidade de sedimentação. Tabela 4 Características dos pacientes com aneurismas das artérias coronárias Variáveis Paciente 1 Paciente 2 Paciente 3 Paciente 4 Paciente 5 Idade de diagnóstico (meses) 12 60 108 63 4 Sexo masculino Sim Sim Sim Sim Sim Dias de febre 7 6 8 9 14 DK clássico Sim Sim Sim Sim Não Dia de febre na 1ᵃ dose IGIV 7 6 4 6 14 Resistência ao tratamento IGIV Não Não Sim Sim NA Dia de febre na 2ᵃ dose IGIV NA NA 6 8 NA MPDN (30mg/kg/dia) Com 1ᵃ dose IGIV Com 2ᵃ dose IGIV Não NA NA Não NA NA Não Não Sim Sim Não Sim Sim Sim Não Classificação AAC Pequenos Pequenos Pequenos Médios Gigantes Z escore máximo NA 4,46 3,56 6,94 13,81 Artérias atingidas ACD; TC ACD; ACE ACE ACE; ACD ACD; CIR; DAE AAC: aneurismas das artérias coronárias; ACD: artéria coronária direita; ACE: artéria coronária esquerda; CIR: artéria circunflexa; DAE: descendente anterior esquerda; DK: doença de Kawasaki; IGIV: imunoglobulina intravenosa; MPDN: metilprednisolona; NA: não aplicável; TC: tronco comum. Na fase aguda, para além do envolvimento coronário, observou-se derrame pericárdico em 10 casos, insuficiência valvular mitral ligeira em três e disfunção ventricular em três, um dos quais com choque cardiogênico. Um apresentou bloqueio atrioventricular (BAV) de 1ºgrau variável. Após a fase aguda, um paciente manteve envolvimento do sistema de condução e dilatação do ventrículo esquerdo (VE) e um ficou com hipertrofia do VE. Os valores de S, E, valor preditivo positivo (VPP) e valor preditivo negativo (VPN) para os diferentes modelos estão apresentados na tabela 5, sendo apenas incluídos os pacientes a quem foi possível categorizar com alto ou baixo risco. Tabela 5 Sensibilidade, especificidade, valores preditivos positivos e negativos dos diferentes modelos Modelo n S E VPP VPN Kobayasahi 34 63,6% 77,3% 53,8% 81% Egami 39 66,7% 73,1% 50% 82,6% Sano 25 28,6% 94,1% 66,7% 77,3% E: especificidade; n: número de casos incluídos; S: sensibilidade; VPN: valor preditivo negativo; VPP: valor preditivo positivo. Discussão A DK é uma vasculite que, embora não tendo uma incidência tão elevada como no Japão, é, ainda assim uma causa importante de doença em idade pediátrica na nossa população. Um diagnóstico e introdução de terapêutica precoces constituem dois fatores muito importantes para reduzir o risco de envolvimento cardíaco. O presente estudo revelou uma percentagem de casos resistentes à IGIV coincidentes com os 10 a 20% descritos na literatura.2 Ao longo dos anos têm sido desenvolvidos esforços no sentido de encontrar fatores clínicos e laboratoriais que possam prever esta resistência de modo a introduzir mais precocemente terapêuticas coadjuvantes. Existem diversos parâmetros descritos na literatura tais como idade, albumina, transaminases, bilirrubina total, neutrófilos, plaquetas, PC-R, VS, entre outros.14,16–20 Neste estudo observou-se que a PC-R e a VS apresentaram capacidade preditora estatisticamente significativa de resistência à IGIV. No caso da PC-R, o ponto de corte ótimo foi de 15,1 mg/dL, com uma sensibilidade de 0,778, especificidade de 0,789 e um OR de 13,125. Pacientes com PC-R superior a 15,1mg/dL apresentam uma probabilidade de resistência à IGIV cerca de 13 vezes superior aos que têm valores inferiores. Relativamente à VS, o ponto de corte ótimo foi de 90,5 mm/h, com uma sensibilidade de 0,667, especificidade 0,857 e OR 12,000. Pacientes com VS superior a 90,5 mm/h apresentam uma probabilidade de resistência à IGIV cerca de 12 vezes superior aos que têm valores inferiores. Combinando estas duas variáveis independentes obteve-se um modelo estatisticamente significativo (p = 0,042), cujo ponto de corte apresenta uma sensibilidade de 0,833 e especificidade de 0,771. No entanto, a variância explicada pelo modelo é apenas de 25% (Nagelkerke R2 = 0,254), pelo que, embora seja estatisticamente significativo, não pode ser validado, o que se deve em grande parte ao tamanho reduzido da amostra. Todavia, é importante realçar esta tendência no sentido de que as duas variáveis poderão ser importantes para prever a resistência à IGIV, antes da sua infusão. O fato destas variáveis terem sido preditoras de resistência, realça o papel da inflamação exuberante nesta doença, etiologia que é defendida como possível precipitante da resposta imunológica que culmina numa vasculite.21 A etiologia da DK continua incerta, no entanto vários fatores são apontados como predisponentes. Um deles é a imaturidade do sistema imunitário, condição que é apoiada pelo fato de afetar predominantemente crianças com idade inferior a cinco anos. Neste estudo 62,5% dos pacientes pertenciam a esta faixa etária, o que, embora corresponda à maioria da amostra, fica aquém dos 80% descritos na literatura.1 Uma possível explicação para estes resultados é a contribuição genética, sendo as percentagens reportadas baseadas em estudos com variedade étnica alargada, incluindo asiáticos. Verificou-se envolvimento coronário em 25% dos casos, com sete a cumprir critérios de dilatação e cinco de aneurismas das artérias coronárias. Os 10% de incidência de AAC, foram superiores aos 4% reportados para os casos devidamente tratados. Comparando os grupos com e sem envolvimento coronário, verificou-se diferença estatisticamente significativa relativamente ao tempo total de febre (p = 0,038). Este resultado corrobora a ideia da persistência da febre ser deletéria e a necessidade da administração de IGIV preferencialmente até ao 10º dia para evitar sequelas cardíacas.9 O uso de corticosteróide na DK continua a ser tema de debate e controvérsia. O mais consensual é usar metilprednisolona por via intravenosa (MPDN iv) na dose de 15 a 30mg/kg/dia durante três dias.9 A MPDN iv, nos pacientes com DK refratária à IGIV, suprime os níveis de citocinas inflamatórias mais rapidamente que uma 2ᵃ dose de IGIV,9 no entanto não está recomendada como primeira linha. Sleeper et al.10 avaliaram o uso de corticosteróide em diferentes momentos, observando-se diferença estatisticamente significativa no aparecimento de AAC apenas nos pacientes refratários à IGIV e que levaram a administração de segunda dose. Neste estudo também se avaliaram complicações cardíacas e achados ecográficos que não o envolvimento coronário. Das complicações cardíacas em fase aguda mais graves destacaram-se três casos de disfunção ventricular esquerda, um com choque cardiogénico e um BAV de 1º grau, complicações essas também descritas na literatura.2,7 Na fase aguda, foi ainda possível observar 10 pacientes com derrame pericárdico sem compromisso hemodinâmico e três com insuficiência valvular mitral ligeira. Em Paris, Chbeir et al.22 obtiveram relação estatisticamente significativa entre resistência à IGIV, AAC e achados na ecografia cardíaca inicial, tais como derrame pericárdico, hiperecogenicidade das coronárias e dilatação coronária. Os autores desse estudo não consideraram a hiperecogenicidade e o afunilamento das coronárias como fatores relevantes, visto que são achados subjetivos, pouco reprodutíveis e podem ser encontrados tanto em doenças febris como em crianças saudáveis.23 Na fase crônica, um paciente manteve envolvimento do sistema de condução e dilatação do VE e outro ficou com hipertrofia do VE. As repercussões cardíacas a longo prazo na DK estão ainda pouco esclarecidas. Friedman et al.6 mostraram maior ocorrência de efeitos adversos cardíacos a longo prazo, tais como morte, transplante cardíaco, cirurgias de bypass coronário e angioplastia primária em pacientes com DK que desenvolveram AAC com escores z superiores e que foram resistentes à IGIV. Um estudo realizado por Holve et al.8 revelou baixa incidência de efeitos adversos cardíacos até os 21 anos de idade, mas uma maior probabilidade de desenvolver hipertensão arterial após os 15 anos de idade. Os modelos validados no Japão apresentaram fraca utilidade clínica no presente estudo (Tabela 4). O modelo com maior especificidade foi o de Sano, embora com sensibilidade muito baixa e apenas com um pequeno número de casos incluído. O modelo de Egami foi o mais sensível para esta amostra, mas, ainda assim, insuficiente para ser validado. Na base dessa diferença de resultados, pode estar o componente genético. Aliás esses resultados vão ao encontro de outros trabalhos realizados fora do Japão, em que nenhum deles conseguiu validar os modelos nas suas amostras.10,14–17,20 É necessário levar em consideração ainda que existiram algumas diferenças no desenho do estudo relativamente aos modelos japoneses, nomeadamente aplicados apenas a pacientes com DK clássica. Um estudo japonês falhou em validar os modelos em uma amostra de pacientes apenas com DK atípica.24 O modelo de Kobayashi foi validado na população japonesa com uma sensibilidade de 0,86 e especificidade de 0,67.11 Ao contrário do presente estudo, a IGIV foi administrada na dose de 1 g/kg em dois dias consecutivos e foi considerada resistência se febre persistente após 24 horas de início de terapêutica, ou se recrudescência com sintomas após período apirético. O modelo de Egami foi validado com uma sensibilidade de 0,78 e especificidade de 0,76; no entanto, foi definida resistência se ausência de diminuição do valor de PC-R em mais de 50% e persistência da febre 48 horas após administração de IGIV.12 Loomba et al.25 não conseguiram validar o modelo de Egami, mesmo aplicando-o separadamente à DK clássica, atípica e por etnias. O modelo de Sano, validado com uma sensibilidade de 0,77 e especificidade de 0,86, foi o único dos três a ajustar o tamanho dos AAC à superfície corporal.13 No entanto, também usou administração de IGIV na dose de 1 g/kg em 2 dias consecutivos e definiu resistência se persistência de febre após 24 horas do término da terapêutica. Este trabalho apresenta as limitações inerentes ao fato de ser um estudo retrospectivo e com uma amostra de tamanho reduzido. Conclusão A PC-R e a VS são variáveis independentes que mostraram tendência preditora de resistência à IGIV com pontos de corte ótimos de 15,1 mg/dL e 90,5 mm/h, respectivamente. Existe, no entanto, necessidade de um estudo com uma amostra de dimensões adequadas para validar um modelo baseado nesses dois dados analíticos. As complicações cardíacas não se resumem às artérias coronárias, devendo ser mais abrangentes o estudo e o seguimento desses pacientes. Os modelos validados para a população japonesa apresentam utilidade muito limitada na amostra deste estudo, reforçando ainda mais a necessidade e a importância de novas abordagens. Original Article Kawasaki Disease: Predictors of Resistance to Intravenous Immunoglobulin and Cardiac Complications https://orcid.org/0000-0001-7047-4170 Faim Diogo 1 https://orcid.org/0000-0001-7506-0647 Henriques Cláudio 1 Brett Ana 2 Francisco Andreia 1 Rodrigues Fernanda 2 Pires António 1 1 Coimbra Portugal Centro Hospitalar e Universitário de Coimbra EPE - Cardiologia Pediátrica Coimbra - Portugal 2 Coimbra Portugal Centro Hospitalar e Universitário de Coimbra EPE - Urgência e Unidade de Infeciologia, Coimbra - Portugal Mailing Address: Diogo Faim • Centro Hospitalar e Universitário de Coimbra EPE - Praceta, R. Prof. Mota Pinto, 3075 Coimbra 3000-075 – Portugal. E-mail: diogofaim92@gmail.com Abstract Background: Kawasaki disease (KD) is the leading cause of acquired cardiac disease in children, in developed countries. Objectives: To identify predictive factors for resistance to intravenous immunoglobulin (IVIG), calculate the effectiveness of Japanese predictive models and characterize cardiac complications. Methods: Retrospective analysis of KD cases admitted in a Portuguese paediatric hospital between january 2006 and july 2018. ROC curves were used to determine predictive factors for resistance and the multivariate logistic regression analysis was used to develop the predictive model. A significance level of 5% was used. Results: 48 patients with a median age of 36 months were included. The IVIG resistance was 21%. Echocardiographic anomalies were noted in 46%, with coronary involvement in 25% of the sample population. As predictive variable of resistance, the C-reactive protein (CRP) presented an AUC ROC = 0.789, optimal cut-off value 15.1 mg/dL, sensitivity (Sn) 77.8% and specificity (Sp) 78.9%. The erythrocyte sedimentation rate (ESR) presented an AUC ROC = 0.781, optimal cut-off value 90.5 mm/h, Sn 66.7% and Sp 85.7%. The model with the two variables showed p = 0.042 and AUC ROC = 0.790. Predictive strength of Japanese models were: Kobayashi (Sn 63.6%, Sp 77.3%), Egami (Sn 66.7%, Sp 73.1%), Sano (Sn 28.6%, Sp 94.1%). Conclusion: CRP and ESR are independent variables that were related to IVIG resistance, with optimal cut-off points of 15.1 mg/dL and 90.5 mm/h, respectively. About half of the patients had some form of cardiac involvement. The Japanese models appeared to be inadequate in our population. (Arq Bras Cardiol. 2021; 116(3):485-491) Keywords: Kawasaki Disease/complications Mucocutaneous Lymph Node Syndrome/complications Drug Resistance Coronary Artery Disease Immunoglobulin Child Introduction Kawasaki disease (KD) is an acute self-limiting vasculitis, which affects medium-sized vessels and is the leading cause of acquired cardiac disease in pediatric age groups.1 Its etiology remains uncertain, but several factors have been associated to it, namely genetic, environmental, and inflammatory ones.2 Although with a worldwide distribution, its highest prevalence is in Japan, where the incidence is on the rise.3 In Portugal, an epidemiological study carried out in 2017 showed an mean annual incidence of 6.5 per 100,000 children under 5 years of age.4 Based on the 2004 American Pediatric Academy criteria,5 classic KD is considered if fever persists for five days or more and if at least four of five additional clinical criteria are observed: nonexudative bilateral conjunctivitis, alterations of the lips and oral cavity, erythematous rash, changes in the extremities, and cervical lymphadenopathy. If fever lasts for five or more days and only two or three additional criteria are present, it is considered atypical KD, if supported by laboratory and echocardiographic data.2 If not treated within an established period, KD can be complicated by coronary artery aneurysms (CAA) in up to 25% of cases.2 Although coronary artery involvement is the most feared consequence of the disease, other cardiac complications are possible.2,6–8 Treatment with intravenous immunoglobulin (IVIG) in the acute phase administered in the first 10 days of illness reduces the incidence of CAA to 4%.2 IVIG resistance occurs in 10-20% of cases, increasing the likelihood of coronary involvement.2 There are different possible approaches in case of IVIG resistance, such as a second dose of IVIG, corticosteroids and/or monoclonal antibodies.9 No benefit has been described when corticosteroids are used in addition to IVIG in the first instance and this therapeutics is currently reserved for refractory cases.10 In order to identify the cases that could potentially be resistant to treatment with IVIG, and benefit from adjuvant therapies in the initial phase, models based on a scoring system have been developed. Three have been validated in the Japanese population, namely the Kobayashi,11 Egami,12 and Sano scoring systems.13 However, several studies have shown that these models are poor predictors in many western populations.10,14,15 The aim of this study was to identify clinical and laboratory predictive factors regarding resistance to IVIG and coronary artery involvement, and to develop a more suitable predictor model of resistance in this population. Secondary objectives regard characterizing the KD cases admitted to a central pediatric hospital over a period of 13 years, to verify the effectiveness of the Japanese scoring systems in our population sample and to analyze the non-coronary cardiac complications of KD. Methods Sample Retrospective analysis of KD cases admitted to the Hospital Pediátrico – Centro Hospitalar e Universitário de Coimbra (HP-CHUC) diagnosed from 01/01/2006 to 06/30/2018. All patients between 30 days and <18 years with KD and treated with IVIG at diagnosis at HP-CHUC were included in the study. All the patients transferred from outlying centers with a diagnosis of KD and managed at these institutions were excluded. The diagnosis of typical and atypical KD was based on the American Academy of Pediatrics criteria. We considered day one of fever on the day the fever started, defined as axillar temperature ≥ 38ºC. Resistance to IVIG was considered if the fever persisted 36 hours after its administration. All patients who received corticosteroids simultaneously with the first dose of IVIG were excluded from the resistance quantification. Dallaire z scores were used to classify the coronary artery morphology defining: coronary artery ectasia if z score between 2 and 2.4, small aneurysm if z score between 2.5 and 4.9, medium aneurysm if z score between 5 and 9.9 and absolute dimension < 8 mm, and giant aneurysm if z score ≥ 10 or absolute dimension ≥ 8 mm. If unable to calculate the z score, the absolute dimensions were used, being small aneurysm if ≥ 2.5 mm and < 4mm, medium aneurysm if ≥ 4 mm and < 8 mm and giant aneurysm if ≥ 8 mm. Regarding other cardiac complications, the coronary artery hyperechogenicity and lack of tapering on echocardiography were not considered as echocardiographic diagnostic criteria. To calculate the effectiveness of the Japanese models, all patients who did not have the necessary data to be considered as high or low risk of IVIG resistance were excluded. Scoring and categorization in high or low risk patients were performed as shown in Table 1. Table 1 Scoring system of the Japanese modelss Model Score High risk Kobayashi ≥ 4 points AST > 100 U/L 2 Na ≤ 133 mmol/L 2 IVIG with fever ≤ 4 days 2 Neutrophils/Leucocytes ≥ 80% 2 CRP ≥ 10 mg/dL 1 Age ≤ 1 year old 1 Platelets ≤ 300,000/µL 1 Egami ≥ 3 points ALT ≥ 80 U/L 2 IVIG with fever ≤ 4 days 1 CRP ≥ 8 mg/dL 1 Age ≤ 6 months 1 Platelets ≤ 300,000/µL 1 Sano ≥ 2 points AST > 200 U/L 1 Total bilirubin ≥ 0.9 mg/dL 1 CRP ≥ 7 mg/dL 1 AST: aspartate transaminase; U: international unity; L: liter; Na: serum sodium; mmol: millimole; IVIG: intravenous immunoglobulin; CRP: C-reactive protein; mg: milligram; dL: deciliter; µL: microliter; ALT: alanine transaminase. Statistical Analysis The SPSS® (IBM®, SPSS® Statistics Inc., Chicago) program version 25.0 was used the performed the statistical analysis. The Shapiro-Wilk test was used to test the normality of the variables. The continuous variables with normal distribution were described using mean and standard deviation (SD) and continuous variables without normal distribution were described using median and interquartile range (IQR). We used the Fisher’s exact test to compare categorical variables, the Student’s t-test to compare parametric variables and the Mann-Whitney test to compare the non-parametric ones. The Receiver Operating Characteristic (ROC) curves were used to evaluate the individual discriminative capacity of each variable and to identify the optimal cutoff points to predict resistance to IVIG. The variables were considered as good predictors if the area under the curve (AUC) > 0.75. Multivariate logistic regression analysis was used to develop the predictive resistance model. A significance level of 5% was used. Results Forty-eight patients met the KD criteria, of whom 32 (66.7%) were male. The median age was 36 months (IQR 16.75-89.25), 62.5% of patients were less than five years old and 10.4% over nine years old. On the day of admission, all the patients presented with fever, with a median duration of five days (IQR 4-8), minimum of one day and maximum of 14 days. Among the five main clinical criteria, nonexudative bilateral conjunctivitis was observed in 94% of cases, alterations of the lips and oral cavity in 90%, erythematous rash in 84%, changes in extremities in 75%, and cervical lymphadenopathy in 69%. The most common findings among oral alterations were cheilitis (67%) and lip erythema (67%), followed by erythema of the oropharynx (50%) and the strawberry tongue (48%). The most prevalent changes in the extremities were erythema (52%), followed by swelling (31%) and peeling (25%). Inflammatory signs at the Bacillus Calmette-Guérin (BCG) vaccination site were observed in 23% of patients. Atypical KD was diagnosed in 17% of the cases. The median duration of hospital stay was two days (IQR 1-6.75). During the acute phase, IVIG 2g/kg was administered to all patients. The median day of illness of the administration was 6.5 (IQR 5-8). Simultaneously, 45-100mg/kg of acetylsalicylic acid (ASA) were administered to 47 patients in the acute phase. Five children received corticosteroids together with the first dose of IVIG. After the acute phase, all patients were medicated with ASA 3-5 mg/kg/day, three patients with clopidogrel and one with enoxaparin. Nine patients were resistant to the IVIG (21%), of which one had atypical KD (p = 0.543). All nine repeated IVIG administration, five of which with methylprednisolone 30 mg/kg/day. Among the variables evaluated as predictors of IVIG resistance (Table 2), C-reactive protein (CRP) presented an AUC ROC of 0.78 (95% confidence interval (CI): 0.632 – 0.947), and the erythrocyte sedimentation rate (ESR), an AUC ROC of 0.781 (95%CI: 0.585 – 0.977). The optimal cut-off value for CRP was 15.1 mg/dL with sensitivity (Sn) of 77.8% and specificity (Sp) of 78.9% (Odds ratio (OR) = 13.125 95%CI: 2.271 – 75.858). The optimal cut-off value for ESR was 90.5 mm/h, with Sn of 66.7% and Sp of 85.7% (OR = 12.000 95%CI: 1.718 – 83.803). A logistic model was developed with these two variables, with a p-value of 0.042, AUC ROC of 0.79 (95%CI: 0.589 – 0.992), with Sn of 83.3% and Sp of 77.1%, but with a 25% variance (Nagelkerke R2 = 0.254). Table 2 Receiver operating characteristic analysis of several variables to predict resistance to intravenous hemoglobulin Characteristic AUC [95%CI] Age 0.542 [0.377; 0.708] IVIG administration day 0.595 [0.403; 0.787] Hemoglobin 0.611 [0.416; 0.806] Leucocytes 0.525 [0.331; 0.719] Neutrophils 0.637 [0.447; 0.828] Platelets 0.513 [0.295; 0.732] ESR 0.781 [0.585; 0.977] CRP 0.789 [0.632; 0.947] Na 0.715 [0.475; 0.955] AST 0.648 [0.434; 0.862] ALT 0.693 [0.486; 0.901] Total bilirubin 0.500 [0,139; 0.861] Albumin 0.693 [0.459; 0.928] AUC: area under the curve; CI: confidence interval; IVIG: intravenous immunoglobulin; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; Na: serum sodium; AST: aspartate transaminase; ALT: alanine transaminase. Coronary artery changes were found in 12 children (25%), seven with ectasia and five with CAA. The comparison between groups with and without coronary artery involvement is shown in Table 3. The duration of fever and the use of corticosteroids were the only significant differences between these two groups. Patients with coronary artery involvement had longer duration of fever (p = 0.038) and greater need for corticotherapy (p = 0.009). Four patients had CAA when methylprednisolone was started. Among the five patients with CAA, three met the criteria for small aneurysms, one for medium aneurysms and one for giant ones. These patients are summarized in Table 4. Table 3 Characteristics of groups with and without coronary artery involvement Characteristics Without coronary involvement (n =36) Coronary involvement (n= 12) p - value Corticoid (n=10) n 4 6 0.009 Resistance to IVIG (n=9) n 5 4 0.173 Age (months) Mean ± sd 59.7 ± 57 47.5 ± 30.1 0.35 Fever duration (days) Mean ± sd 7.4 ± 2.8 9.4 ± 3 0.038 IVIG administration day Mean ± sd 6.5 ± 2.9 7.6 ± 3.5 0.283 Atypical KD n 6 2 0.686 Haemoglobin (g/dL) Mean ± sd 11.5 ± 1.3 11.2 ± 1.3 0.359 Leucocytes (/µL) Mean ± sd 14,174 ± 6,010 15,216 ± 6,918 0.619 Neutrophils (/µL) Mean ± sd 9,515 ± 4,770 10,994 ± 5,629 0.378 Platelets (/µL) Mean ± sd 328,389 ± 127,125 362,667 ± 282,652 0.691 CRP (mg/dL) Mean ± sd 10.4 ± 8.8 19.6 ±25 0.243 ESR (mm/h) Mean ± sd 71.6 ± 19 76.7 ± 33.4 0.672 Na (mmol/L) Mean ± sd 137 ± 4 137 ± 5 0.869 AST (U/L) Mean ± sd 64 ± 53 101 ± 91 0.222 ALT (U/L) Mean ± sd 99 ± 116 113 ± 86 0.712 Total bilirubin (mg/dL) Mean ± sd 1.9 ± 2 2.8 ± 3 0.538 Albumin (g/L) Mean ± sd 35.6 ± 4.3 34.9 ± 7.2 0.77 n: absolute value; IVIG: intravenous immunoglobulin; sd: standard deviation; KD: Kawasaki disease; g: gram; dL: deciliter; µL: microliter; CRP: C-reactive protein; mg: milligram; ESR: erythrocyte sedimentation rate; h: hour; Na: serum sodium; mmol: millimole; L: litre; AST: aspartate transaminase; U: international unity; ALT: alanine transaminase. Table 4 Characteristics of patients with coronary artery aneurysms Characteristics Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Age (months) 12 60 108 63 4 Male Yes Yes Yes Yes Yes Fever duration (days) 7 6 8 9 14 Classic KD Yes Yes Yes Yes No Day of fever in IVIG 1st dose 7 6 4 6 14 Resistance to IVIG No No Yes Yes NA Day of fever in IVIG 2nd dose NA NA 6 8 NA MPDN (30mg/Kg/day) With IVIG 1st dose With IVIG 2nd dose No NA NA No NA NA Yes No Yes Yes No Yes Yes Yes No CAA classification Small Small Small Medium Giant Maximum z score NA 4.46 3.56 6.94 13.81 Arteries involved RCA; CT RCA; LCA LCA RCA; LCA RCA; LAD; LCX KD: Kawasaki disease; IVIG: intravenous immunoglobulin; MPDN: methylprednisolone; mg: milligram; Kg: kilogram; CAA: coronary artery aneurysms; RCA: right coronary artery, CT: common trunk; LCA: left coronary artery; LAD: left anterior descending artery; NA: not applicable; LCX: left circumflex artery. In the acute phase, in addition to coronary involvement, 10 patients presented with pericardial effusion, three with mild mitral valve regurgitation, two with left ventricular systolic dysfunction, one with cardiogenic shock and one with variable first degree atrioventricular (AV) block. After the acute phase, the patient with the AV block developed left ventricle (LV) dilation and another patient developed LV hypertrophy. Table 5 summarizes Sn, Sp,and the positive (PPV) and negative predictive values (NPV) for the Japanese models in our sample. Table 5 Statistical values of the Japanese models in our study Model n Sn (%) Sp (%) PPV (%) NPV (%) Kobayasahi 34 63.6 77.3 53.8 81 Egami 39 66.7 73.1 50 82.6 Sano 25 28.6 94.1 66.7 77.3 n: absolute value; Sn: sensitivity; Sp: Specificity; PPV: positive predictive value; NPV: negative predictive value. Discussion Despite the lower incidence compared to Japan, KD is a vasculitis that is still an important cause of pediatric disease in our population. Early diagnosis and management are two important factors that appear to reduce cardiac involvement. Our study revealed an incidence of IVIG resistance similar to the 10 to 20% described in the literature.2 Over the years, efforts have been made to find clinical and laboratory factors that can predict this resistance in order to introduce adjuvant therapies at an early stage of the disease. There are several parameters in the literature that have been studied for this purpose, such as age, serum albumin, transaminases, total bilirubin, neutrophils count, platelet count, CRP, ESR, among others.14,16–20 In our study, CRP and ESR presented a statically significant predictive capacity in relation to IVIG resistance. For CRP, the optimal cut-off point was 15.1 mg/dL (Sn 77.8%, Sp 78.9%, OR 13.125). Patients with CRP values above 15.1 mg/dL are about 13 times more likely to be resistant to IVIG than those with lower values. Concerning ESR, the optimal cut-off point was 90.5 mm/h (Sn 66.7%, Sp 85.7%, OR 12.000). Patients with ESR greater than 90.5 mm/h have a probability of resistance to IVIG approximately 12 times higher than those with lower values. Combining these two independent variables, a statistically significant model was obtained (p = 0.042), whose cut-off point has Sn of 83.3% and Sp of 77.1%. Despite these encouraging results, the variance explained by the model is only 25% (Nagelkerke R2 = 0.254). Thus, although statistically significant, it cannot be validated, which is largely due to the small sample size. Nevertheless, based on these trends, the base-line values for CRP and ESR should be known prior to IVIG administration. The resistance predictor capacity highlights the role of inflammation in this disease, a possible underlying trigger in KD vasculitis.21 The etiology of KD remains uncertain, however, predisposing factors have been put forward. One, is the immaturity of the immune system, a theory that is supported by the fact that KD predominantly affects children under the age of five. In our study, 62.5% of the patients belonged to this age group, which, although corresponding to the majority of the sample, is below the 80% described in the literature.1 A possible explanation for this result is genetic contribution, since the incidences described in the literature are from studies with a wide range of ethnicities, including Asian children. Coronary artery involvement occurred in 12 (25%) children, seven with ectasia and five with CAA. Therefore, the incidence of CAA was 10%, which is higher than the 4% reported in the literature. Comparing the groups with and without coronary artery involvement, a statistically significant difference was found regarding the duration of fever (p = 0.038). This result highlights the deleterious effects of persistent fever and the need for IVIG administration, preferably up to the tenth day of the disease, in order to avoid cardiac sequelae.9 The use of corticosteroids in KD is still a topic of debate and controversy. The most consensual is the use intravenous methylprednisolone (MPDN) at a dose of 15 to 30 mg/kg/day, for three days.9 In patients with refractory KD, MPDN supresses the inflammatory cytokine levels more quickly than a second dose of IVIG,9 although it is not recommended as a first-line treatment. Sleeper et al.10 evaluated the impact of corticosteroids at different times of the disease and showed that, with regards to the development of CAA, the only statistically significant difference was in those refractory to the first dose of IVIG which combined corticosteroids with the second dose of IVIG. The cardiac complications and echocardiographic findings, others than coronary artery involvement were also evaluated. Three cases of left ventricular systolic dysfunction were identified, one of which with cardiogenic shock, a complication also described in the literature.2,7 In the acute phase, ten patients presented with pericardial effusion without hemodynamic compromise, three patients with mild mitral valve regurgitation and one with first degree AV block. Chbeir et al.22 found a relation between resistance to IVIG, CAA, and initial cardiac echocardiographic findings such as pericardial effusion, coronary hyperechogenicity, and coronary ectasia. The coronary hyperechogenicity and the lack of tapering on echocardiography were not considered as relevant factors, since they are subjective findings, poorly reproducible and can be found both in febrile illnesses and in healthy children.23 During the chronic phase, one patient remained with conduction system impairment and developed LV dilation, and another patient developed LV hypertrophy. The long-term cardiac repercussions in KD remain unclear. Friedman et al.6 reported an increase in the occurrence of long-term adverse cardiac effects, leading to primary angioplasty, coronary bypass surgery, heart transplantation, and death, in patients who developed CAA with higher z scores and who were initially resistant to IVIG. A study by Holve et al.8 revealed a low incidence of adverse cardiac effects in subjects up to 21 years of age, but a greater likelihood of developing high blood pressure from the age of 15. Japanese predictive models presented poor clinical utility in this study (Table 4). The model with the highest specificity was Sano’s, although with very low sensitivity and with only a small number of cases included. Egami’s model was the most sensitive, however, not powerful enough to be validated. The genetic component may be the explanation for these differences. In fact, the results presented here are similar to those of other studies carried out outside Japan, in which none were able to validate the models in their samples.10,14–17,20 It is still important to note the differences in the study design in relation to the Japanese models, namely that they were applied only to patients with classical KD. Another Japanese study failed to validate the models in a sample exclusively composed of atypical KD cases.24 The Kobayashi model was validated for the Japanese population with Sn of 86% and Sp of 67%.11 Contrary to the present study, IVIG was administered at a dose of 1 g/kg on two consecutive days and resistance was considered if fever persisted 24 hours after the beginning of the treatment, or in case of recurrence after a period without fever. The Egami model was validated with Sn of 78% and Sp of 76%, however, resistance was considered if the CRP value did not decrease by more than 50% and fever persisted for longer than 48 hours after IVIG administration.12 Loomba et al.25 were not able to validate the Egami model even when applying it separately to classical and atypical KD, and by ethnicity. The Sano model, validated with Sn of 77% and Sp of 86%, was the only one of the three to adjust the size of the CAA to the body surface.13 However, it also used IVIG at a dose of 1 g/kg on two consecutive days and defined resistance if fever persisted 24 hours after the end of therapy. The main limitations of this analysis are related to its retrospective methodology and sample size. Conclusions CRP and ESR are independent variables that showed a predictive trend regarding resistance to IVIG, with optimal cut-off values of 15.1 mg/dL and 90.5 mm/h, respectively. However, there is a need for a multicenter study with a sample of adequate dimensions to validate a model based on these two analytical parameters. Cardiac complications are not limited to coronary arteries, and the study and follow-up of these patients should be more widespread. The validated models for the Japanese population have very limited utility in our population, further reinforcing the need and importance of new approaches. Fontes de Financiamento O presente estudo não contou com fontes de financiamento externas. Vinculação Acadêmica Não há vinculação deste estudo a programas de pós-graduação. Aprovação Ética e Consentimento Informado Este artigo não contém estudos com humanos ou animais realizados por nenhum dos autores. Author Contributions Conception and design of the research: Faim D, Henriques C, Brett A, Francisco A, Rodrigues F, Pires A; Data acquisition: Faim D, Henriques C; Analysis and interpretation of the data: Faim D, Brett A, Francisco A; Statistical analysis and Writing of the manuscript: Faim D; Critical revision of the manuscript for intellectual content: Faim D, Brett A, Francisco A, Rodrigues F, Pires A. Potential Conflict of Interest The authors report no conflict of interest concerning the materials and methods used in this study or the findings specified in this paper. Sources of Funding There was no external funding source for this study. Study Association This study is not associated with any thesis or dissertation. Ethics Approval and Consent to Participate This article does not contain any studies with human participants or animals performed by any of the authors.	2 GRAM PER KILOGRAM	DrugDosageText	CC BY	33470332	19,012,502	2021-03
What was the dosage of drug 'METHYLPREDNISOLONE'?	Kawasaki Disease: Predictors of Resistance to Intravenous Immunoglobulin and Cardiac Complications. Kawasaki disease (KD) is the leading cause of acquired cardiac disease in children, in developed countries. To identify predictive factors for resistance to intravenous immunoglobulin (IVIG), calculate the effectiveness of Japanese predictive models and characterize cardiac complications. Retrospective analysis of KD cases admitted in a Portuguese paediatric hospital between january 2006 and july 2018. ROC curves were used to determine predictive factors for resistance and the multivariate logistic regression analysis was used to develop the predictive model. A significance level of 5% was used. 48 patients with a median age of 36 months were included. The IVIG resistance was 21%. Echocardiographic anomalies were noted in 46%, with coronary involvement in 25% of the sample population. As predictive variable of resistance, the C-reactive protein (CRP) presented an AUC ROC = 0.789, optimal cut-off value 15.1 mg/dL, sensitivity (Sn) 77.8% and specificity (Sp) 78.9%. The erythrocyte sedimentation rate (ESR) presented an AUC ROC = 0.781, optimal cut-off value 90.5 mm/h, Sn 66.7% and Sp 85.7%. The model with the two variables showed p = 0.042 and AUC ROC = 0.790. Predictive strength of Japanese models were: Kobayashi (Sn 63.6%, Sp 77.3%), Egami (Sn 66.7%, Sp 73.1%), Sano (Sn 28.6%, Sp 94.1%). CRP and ESR are independent variables that were related to IVIG resistance, with optimal cut-off points of 15.1 mg/dL and 90.5 mm/h, respectively. About half of the patients had some form of cardiac involvement. The Japanese models appeared to be inadequate in our population. (Arq Bras Cardiol. 2021; 116(3):485-491). Introdução A doença de Kawasaki (DK) é uma vasculite aguda e autolimitada que afeta os vasos de média dimensão, sendo a principal causa de cardiopatia adquirida em idade pediátrica.1 A sua etiologia continua incerta, mas vários fatores têm sido associados à doença, nomeadamente genéticos, ambientais e inflamatórios.2 Embora com distribuição mundial, a sua maior prevalência é no Japão, com incidência crescente.3 Em Portugal, um estudo epidemiológico realizado em 2017 descreveu uma incidência anual média de 6,5 por 100.000 crianças com menos de 5 anos de idade.4 Com base nos critérios da American Pediatric Academy de 2004,5 considera-se DK clássica se febre ≥ 5 dias e presença de pelo menos 4 de 5 critérios clínicos adicionais: conjuntivite bilateral não exsudativa, alterações dos lábios e da mucosa oral, exantema polimorfo, alterações das extremidades e linfadenopatia cervical não supurativa. Se febre ≥ 5 dias e apenas 2 ou 3 critérios adicionais, considera-se DK atípica e é necessário recorrer a dados laboratoriais e ecográficos para melhor sustentar o diagnóstico.2 Se não tratada atempadamente, a DK pode complicar-se com aneurismas das artérias coronárias (AAC) em até 25% dos casos.2 Apesar de o envolvimento coronário ser a consequência mais temida da doença, são possíveis outras complicações cardíacas.2,6–8 O tratamento com imunoglobulina intravenosa (IGIV) na fase aguda continua a ser a principal terapêutica e, se administrada nos primeiros 10 dias de doença, diminui a incidência de AAC para 4%.2 A resistência à IGIV ocorre em 10% a 20% dos casos, aumentado a probabilidade de envolvimento coronário.2 Há diferentes abordagens possíveis quando há resistência, nomeadamente uma segunda dose de IGIV, corticosteroides e/ou anticorpos monoclonais.9 Não há descrição de benefício na administração inicial de corticosteroide juntamente com a IGIV a todos os pacientes, sendo atualmente tal terapêutica reservada para os casos refratários.10 Com o objetivo de identificar os casos que potencialmente poderiam ser resistentes ao tratamento com IGIV, e como tal beneficiar de outras terapêuticas na fase inicial, foram desenvolvidos modelos com base em um sistema de escores, nomeadamente os de Kobayashi,11 Egami12 e Sano,13 que foram validados para a população japonesa. No entanto, diversos estudos mostraram que esses modelos são fracos preditores em diversas populações ocidentais.10,14,15 O objetivo desse estudo foi identificar fatores preditores clínicos e analíticos de resistência à IGIV e de envolvimento coronário e construir um modelo preditor de resistência mais adequado para essa população. Como objetivos adicionais, pretendeu-se caracterizar os casos de DK em um hospital pediátrico de nível III nos últimos anos, calcular a eficácia dos escores japoneses nessa amostra e analisar as complicações cardíacas não coronárias da DK. Métodos Amostra Estudo retrospectivo dos casos diagnosticados com DK desde 1º de janeiro de 2006 a 30 de junho de 2018 no Hospital Pediátrico – Centro Hospitalar e Universitário de Coimbra (HP-CHUC). Foram incluídas todas as crianças e adolescentes com idades entre 30 dias e < 18 anos, com diagnóstico e terapêutica de fase aguda realizados no HP-CHUC. Foram excluídos todos os pacientes transferidos para o HP-CHUC já com diagnóstico e/ou terapêutica realizados em outro hospital. Foram utilizados os critérios da American Academy of Pediatrics para diagnóstico de DK clássica e de DK atípica. Considerou-se D1 de febre o dia inaugural de febre, definida como temperatura axilar ≥ 38°C. Considerou-se resistência à IGIV se a febre persistiu 36 horas após a sua administração, tendo sido excluídos todos os casos em que foi administrado corticosteroide concomitantemente com a primeira dose de IGIV. Para classificar o envolvimento coronário, foram usados os escores z (desvios padrões) de Dallaire, definindo-se dilatação se escore z entre 2 e 2,4, aneurisma pequeno se escore z entre 2,5 e 4,9, aneurisma médio se entre 5 e 9,9 e dimensão absoluta inferior a 8 mm, aneurisma gigante se escore z ≥ 10 ou dimensão absoluta ≥ 8 mm. Nos casos em que a informação foi insuficiente para se calcular o escore z, utilizaram-se os valores absolutos, sendo aneurisma pequeno se ≥ 2,5mm e < 4mm, médio se ≥ 4mm e < 8mm e gigante se ≥ 8 mm. Nas complicações cardíacas, os achados ecocardiográficos de hiperecogenicidade e afunilamento das artérias coronárias não foram considerados. Para calcular a eficácia dos modelos, foram excluídos todos os pacientes que não apresentavam os dados necessários para serem considerados como alto ou baixo risco de resistência para determinado modelo. A pontuação e a categorização em pacientes de alto ou baixo risco foram realizadas conforme evidenciado na Tabela 1. Tabela 1 Sistema de pontuação dos modelos avaliados Escore Pontos Alto risco Kobayashi ≥ 4 pontos AST > 100 U/L 2 Na ≤ 133 mmol/L 2 IGIV com febre ≤ D4 2 Neutrófilos/Leucócitos ≥ 80% 2 PC-R ≥ 10 mg/dL 1 Idade ≤ 1 ano 1 Plaquetas ≤ 300.000/uL 1 Egami ≥ 3 pontos ALT ≥ 80 U/L 2 IGIV com febre ≤ D4 1 PC-R ≥ 8 mg/dL 1 Idade ≤ 6 meses 1 Plaquetas ≤ 300.000/uL 1 Sano ≥ 2 pontos AST > 200 U/L 1 Bilirrubina total ≥ 0,9 mg/dL 1 PC-R ≥ 7 mg/dL 1 ALT: alanina aminotransferase; AST: aspartato aminotransferase; dL: decilitro; L: litro; mg: miligrama; IGIV: imunoglobulina intravenosa; mmol: milimole; Na: sódio plasmático; PC-R: proteína C-reativa; U: unidade internacional; uL: microlitro. Análise Estatística Análise estatística efetuada com recurso ao programa SPSS® (IBM®, SPSS® Statistics Inc., Chicago), versão 25.0. Para testar a normalidade da amostra, foi utilizado o teste de Shapiro-Wilk. As variáveis contínuas com distribuição normal foram descritas por meio de média e desvio padrão, as variáveis contínuas sem distribuição normal foram descritas através de mediana e amplitude interquartil (AIQ). Para comparação de variáveis categóricas, foi utilizado o teste exato de Fisher; das variáveis numéricas paramétricas, o teste de t-student não pareado; e das variáveis numéricas não paramétricas, o teste de Mann-Whitney. Foram construídas curvas receiver operating characteristic (ROC) para avaliar a capacidade discriminativa individual de cada variável e identificar pontos de corte para a predição de resistência à IGIV. As variáveis foram consideradas preditoras se com uma area under the curve (AUC) superior a 0,75. Para a elaboração de um modelo preditor de resistência, foi utilizada a regressão logística multivariada. O nível de significância utilizado nesse estudo foi de 5%. Resultados Cumpriram critérios de DK 48 pacientes, sendo 32 (66,7%) do sexo masculino. A mediana de idades foi de 36 meses (AIQ 16,75-89,25), com 62,5% abaixo dos 5 anos de idade e 10,4% acima dos 9 anos. No dia de admissão, a totalidade dos pacientes apresentava-se com febre, com uma mediana de 5 dias de febre (AIQ 4-8), mínimo de 1 e máximo de 14 dias. Dos cinco critérios clínicos principais, observou-se conjuntivite não purulenta em 94%, alterações orais em 90%, exantema em 84%, alterações das extremidades em 75% e linfadenopatia cervical em 69%. Dentre as alterações orais, as mais prevalentes foram a queilite (67%) e o eritema labial (67%), seguidos de eritema da orofaringe (50%) e língua em framboesa (48%). Entre as alterações das extremidades, a mais prevalente foi eritema (52%), seguido de edema duro (31%) e descamação (25%). Os sinais inflamatórios no local de inoculação da vacina Bacillus Calmette-Guérin (BCG) foram descritos em 23%. Foi diagnosticada DK atípica em 17% dos casos. A mediana de dias de internamento foi de 2 dias (AIQ 1-6,75). A totalidade dos pacientes foi medicada em fase aguda com IGIV 2 g/kg com mediana do dia de administração de 6,5 dias (AIQ 5-8). Foram medicados em fase aguda 47 pacientes com ácido acetilsalicílico (AAS) em doses entre 45 a 100mg/kg/dia. Cinco pacientes foram ainda medicados com corticosteroide juntamente com a primeira dose de IGIV. Após a fase aguda, todos os pacientes foram medicados com AAS em dose de 3 a 5 mg/kg/dia, três pacientes com clopidogrel e um com enoxaparina. Verificou-se resistência à IGIV em nove casos (21%), dos quais havia um caso de DK atípica (p = 0,543). Dos nove pacientes resistentes à terapêutica, foi administrada uma segunda dose de IGIV 2 g/kg e a cinco destes, metilprednisolona na dose de 30 mg/kg/dia. Entre as diferentes variáveis avaliadas como preditoras de resistência (Tabela 2), a proteína C-reativa (PC-R) apresentou uma AUC ROC de 0,78 (IC95%: 0,632-0,947) e a velocidade de sedimentação (VS) uma AUC ROC de 0,781 (IC95%: 0,585-0,977). O ponto de corte para a PC-R foi de 15,1 mg/dL com uma sensibilidade (S) de 0,778 e especificidade (E) de 0,789 [Odds ratio (OR) = 13,125 IC95%: 2,271-75,858]. O ponto de corte para a VS foi de 90,5 mm/h, verificando-se uma sensibilidade de 0,667 e especificidade de 0,857 (OR = 12,000 IC95%: 1,718-83,803). Foi ajustado um modelo logístico com as duas variáveis PC-R e VS, modelo este que apresentou um valor p de 0,042 e AUC ROC de 0,790 (IC95%: 0,589-0,992). No ponto de corte ótimo, a sensibilidade foi de 0,833 e a especificidade foi de 0,771. Apresentou ainda uma variância de 25% (Nagelkerke R2 = 0,254). Tabela 2 Análise ROC de diversas variáveis para predizer resistência à IGIV Variável AUC [IC 95%] Idade 0,542 [0,377; 0,708] Dia de administração IGIV 0,595 [0,403; 0,787] Hemoglobina 0,611 [0,416; 0,806] Leucócitos 0,525 [0,331; 0,719] Neutrófilos 0,637 [0,447; 0,828] Plaquetas 0,513 [0,295; 0,732] VS 0,781 [0.585; 0,977] PC-R 0,789 [0,632; 0,947] Na 0,715 [0,475; 0,955] AST 0,648 [0,434; 0,862] ALT 0,693 [0,486; 0,901] Bilirrubina total 0,500 [0,139; 0,861] Albumina 0,693 [0,459; 0,928] ALT: alanina aminotransferase; AST: aspartato aminotransferase; AUC: área abaixo da curva; IC: intervalo de confiança; IGIV: imunoglobulina intravenosa; Na: sódio plasmático; PC-R: proteína C-reativa; ROC: receiver operating curve; VS: velocidade de sedimentação. Ocorreu envolvimento coronário em 12 casos (25%). Sete pacientes desenvolveram dilatação das artérias coronárias e cinco, AAC. Na Tabela 3, comparam-se os grupos sem e com envolvimento coronário. Observaram-se diferenças apenas na duração da febre e na utilização de corticosteroides. O tempo total de febre foi mais elevado no grupo com envolvimento coronário (p = 0,038). Esse grupo recebeu mais vezes corticosteroide (p = 0,009). Quatro desses pacientes já tinham diagnóstico de envolvimento coronário prévio à administração desse fármaco. Dos pacientes com AAC, três cumpriram critérios para aneurismas pequenos, um para aneurismas médios e um para aneurismas gigantes. Esses pacientes estão caracterizados na Tabela 4. Tabela 3 Características dos grupos com e sem envolvimento coronário Variáveis Sem envolvimento coronário (n =36) Envolvimento coronário (n = 12) p Corticosteroide (n = 10) N 4 6 0,009 Resistência à IGIV (n = 9) N 5 4 0,173 Idade (meses) Média ± DP 59,7 ± 57 47,5 ± 30,1 0,35 Dia de resolução da febre Média ± DP 7,4 ± 2,8 9,4 ± 3 0,038 Dia de administração de IGIV Média ± DP 6,5 ± 2,9 7,6 ± 3,5 0,283 DK atípica n 6 2 0,686 Hemoglobina (g/dL) Média ± DP 11,5 ± 1,3 11,2 ± 1,3 0,359 Leucócitos (/uL) Média ± DP 14.174 ± 6.010 15.216 ± 6.918 0,619 Neutrófilos (/uL) Média ± DP 9.515 ± 4.770 10.994 ± 5.629 0,378 Plaquetas (/uL) Média ± DP 32.8389 ± 12.7125 36.2667 ± 28.2652 0,691 PC-R (mg/dL) Média ± DP 10,4 ± 8,8 19,6 ±25 0,243 VS (mm/h) Média ± DP 71,6 ± 19 76,7 ± 33,4 0,672 Na (mmol/L) Média ± DP 137 ± 4 137 ± 5 0,869 AST (U/L) Média ± DP 64 ± 53 101 ± 91 0,222 ALT (U/L) Média ± DP 99 ± 116 113 ± 86 0,712 Bilirrubina total (mg/dL) Média ± DP 1,9 ± 2 2,8 ± 3 0,538 Albumina (g/L) Média ± DP 35,6 ± 4,3 34,9 ± 7,2 0,77 AIQ: amplitude interquartil; ALT: alanina aminotransferase; AST: aspartato aminotransferase; dL: decilitro; DK: doença de Kawasaki; DP: desvio padrão; g: grama; h: hora; IGIV: imunoglobulina intravenosa; L: litro; mg: miligrama; mmol: milimole; n: número absoluto; Na: sódio plasmático; p: valor p; PC-R: proteína C-reativa; U: unidade internacional; uL: microlitro; VS: velocidade de sedimentação. Tabela 4 Características dos pacientes com aneurismas das artérias coronárias Variáveis Paciente 1 Paciente 2 Paciente 3 Paciente 4 Paciente 5 Idade de diagnóstico (meses) 12 60 108 63 4 Sexo masculino Sim Sim Sim Sim Sim Dias de febre 7 6 8 9 14 DK clássico Sim Sim Sim Sim Não Dia de febre na 1ᵃ dose IGIV 7 6 4 6 14 Resistência ao tratamento IGIV Não Não Sim Sim NA Dia de febre na 2ᵃ dose IGIV NA NA 6 8 NA MPDN (30mg/kg/dia) Com 1ᵃ dose IGIV Com 2ᵃ dose IGIV Não NA NA Não NA NA Não Não Sim Sim Não Sim Sim Sim Não Classificação AAC Pequenos Pequenos Pequenos Médios Gigantes Z escore máximo NA 4,46 3,56 6,94 13,81 Artérias atingidas ACD; TC ACD; ACE ACE ACE; ACD ACD; CIR; DAE AAC: aneurismas das artérias coronárias; ACD: artéria coronária direita; ACE: artéria coronária esquerda; CIR: artéria circunflexa; DAE: descendente anterior esquerda; DK: doença de Kawasaki; IGIV: imunoglobulina intravenosa; MPDN: metilprednisolona; NA: não aplicável; TC: tronco comum. Na fase aguda, para além do envolvimento coronário, observou-se derrame pericárdico em 10 casos, insuficiência valvular mitral ligeira em três e disfunção ventricular em três, um dos quais com choque cardiogênico. Um apresentou bloqueio atrioventricular (BAV) de 1ºgrau variável. Após a fase aguda, um paciente manteve envolvimento do sistema de condução e dilatação do ventrículo esquerdo (VE) e um ficou com hipertrofia do VE. Os valores de S, E, valor preditivo positivo (VPP) e valor preditivo negativo (VPN) para os diferentes modelos estão apresentados na tabela 5, sendo apenas incluídos os pacientes a quem foi possível categorizar com alto ou baixo risco. Tabela 5 Sensibilidade, especificidade, valores preditivos positivos e negativos dos diferentes modelos Modelo n S E VPP VPN Kobayasahi 34 63,6% 77,3% 53,8% 81% Egami 39 66,7% 73,1% 50% 82,6% Sano 25 28,6% 94,1% 66,7% 77,3% E: especificidade; n: número de casos incluídos; S: sensibilidade; VPN: valor preditivo negativo; VPP: valor preditivo positivo. Discussão A DK é uma vasculite que, embora não tendo uma incidência tão elevada como no Japão, é, ainda assim uma causa importante de doença em idade pediátrica na nossa população. Um diagnóstico e introdução de terapêutica precoces constituem dois fatores muito importantes para reduzir o risco de envolvimento cardíaco. O presente estudo revelou uma percentagem de casos resistentes à IGIV coincidentes com os 10 a 20% descritos na literatura.2 Ao longo dos anos têm sido desenvolvidos esforços no sentido de encontrar fatores clínicos e laboratoriais que possam prever esta resistência de modo a introduzir mais precocemente terapêuticas coadjuvantes. Existem diversos parâmetros descritos na literatura tais como idade, albumina, transaminases, bilirrubina total, neutrófilos, plaquetas, PC-R, VS, entre outros.14,16–20 Neste estudo observou-se que a PC-R e a VS apresentaram capacidade preditora estatisticamente significativa de resistência à IGIV. No caso da PC-R, o ponto de corte ótimo foi de 15,1 mg/dL, com uma sensibilidade de 0,778, especificidade de 0,789 e um OR de 13,125. Pacientes com PC-R superior a 15,1mg/dL apresentam uma probabilidade de resistência à IGIV cerca de 13 vezes superior aos que têm valores inferiores. Relativamente à VS, o ponto de corte ótimo foi de 90,5 mm/h, com uma sensibilidade de 0,667, especificidade 0,857 e OR 12,000. Pacientes com VS superior a 90,5 mm/h apresentam uma probabilidade de resistência à IGIV cerca de 12 vezes superior aos que têm valores inferiores. Combinando estas duas variáveis independentes obteve-se um modelo estatisticamente significativo (p = 0,042), cujo ponto de corte apresenta uma sensibilidade de 0,833 e especificidade de 0,771. No entanto, a variância explicada pelo modelo é apenas de 25% (Nagelkerke R2 = 0,254), pelo que, embora seja estatisticamente significativo, não pode ser validado, o que se deve em grande parte ao tamanho reduzido da amostra. Todavia, é importante realçar esta tendência no sentido de que as duas variáveis poderão ser importantes para prever a resistência à IGIV, antes da sua infusão. O fato destas variáveis terem sido preditoras de resistência, realça o papel da inflamação exuberante nesta doença, etiologia que é defendida como possível precipitante da resposta imunológica que culmina numa vasculite.21 A etiologia da DK continua incerta, no entanto vários fatores são apontados como predisponentes. Um deles é a imaturidade do sistema imunitário, condição que é apoiada pelo fato de afetar predominantemente crianças com idade inferior a cinco anos. Neste estudo 62,5% dos pacientes pertenciam a esta faixa etária, o que, embora corresponda à maioria da amostra, fica aquém dos 80% descritos na literatura.1 Uma possível explicação para estes resultados é a contribuição genética, sendo as percentagens reportadas baseadas em estudos com variedade étnica alargada, incluindo asiáticos. Verificou-se envolvimento coronário em 25% dos casos, com sete a cumprir critérios de dilatação e cinco de aneurismas das artérias coronárias. Os 10% de incidência de AAC, foram superiores aos 4% reportados para os casos devidamente tratados. Comparando os grupos com e sem envolvimento coronário, verificou-se diferença estatisticamente significativa relativamente ao tempo total de febre (p = 0,038). Este resultado corrobora a ideia da persistência da febre ser deletéria e a necessidade da administração de IGIV preferencialmente até ao 10º dia para evitar sequelas cardíacas.9 O uso de corticosteróide na DK continua a ser tema de debate e controvérsia. O mais consensual é usar metilprednisolona por via intravenosa (MPDN iv) na dose de 15 a 30mg/kg/dia durante três dias.9 A MPDN iv, nos pacientes com DK refratária à IGIV, suprime os níveis de citocinas inflamatórias mais rapidamente que uma 2ᵃ dose de IGIV,9 no entanto não está recomendada como primeira linha. Sleeper et al.10 avaliaram o uso de corticosteróide em diferentes momentos, observando-se diferença estatisticamente significativa no aparecimento de AAC apenas nos pacientes refratários à IGIV e que levaram a administração de segunda dose. Neste estudo também se avaliaram complicações cardíacas e achados ecográficos que não o envolvimento coronário. Das complicações cardíacas em fase aguda mais graves destacaram-se três casos de disfunção ventricular esquerda, um com choque cardiogénico e um BAV de 1º grau, complicações essas também descritas na literatura.2,7 Na fase aguda, foi ainda possível observar 10 pacientes com derrame pericárdico sem compromisso hemodinâmico e três com insuficiência valvular mitral ligeira. Em Paris, Chbeir et al.22 obtiveram relação estatisticamente significativa entre resistência à IGIV, AAC e achados na ecografia cardíaca inicial, tais como derrame pericárdico, hiperecogenicidade das coronárias e dilatação coronária. Os autores desse estudo não consideraram a hiperecogenicidade e o afunilamento das coronárias como fatores relevantes, visto que são achados subjetivos, pouco reprodutíveis e podem ser encontrados tanto em doenças febris como em crianças saudáveis.23 Na fase crônica, um paciente manteve envolvimento do sistema de condução e dilatação do VE e outro ficou com hipertrofia do VE. As repercussões cardíacas a longo prazo na DK estão ainda pouco esclarecidas. Friedman et al.6 mostraram maior ocorrência de efeitos adversos cardíacos a longo prazo, tais como morte, transplante cardíaco, cirurgias de bypass coronário e angioplastia primária em pacientes com DK que desenvolveram AAC com escores z superiores e que foram resistentes à IGIV. Um estudo realizado por Holve et al.8 revelou baixa incidência de efeitos adversos cardíacos até os 21 anos de idade, mas uma maior probabilidade de desenvolver hipertensão arterial após os 15 anos de idade. Os modelos validados no Japão apresentaram fraca utilidade clínica no presente estudo (Tabela 4). O modelo com maior especificidade foi o de Sano, embora com sensibilidade muito baixa e apenas com um pequeno número de casos incluído. O modelo de Egami foi o mais sensível para esta amostra, mas, ainda assim, insuficiente para ser validado. Na base dessa diferença de resultados, pode estar o componente genético. Aliás esses resultados vão ao encontro de outros trabalhos realizados fora do Japão, em que nenhum deles conseguiu validar os modelos nas suas amostras.10,14–17,20 É necessário levar em consideração ainda que existiram algumas diferenças no desenho do estudo relativamente aos modelos japoneses, nomeadamente aplicados apenas a pacientes com DK clássica. Um estudo japonês falhou em validar os modelos em uma amostra de pacientes apenas com DK atípica.24 O modelo de Kobayashi foi validado na população japonesa com uma sensibilidade de 0,86 e especificidade de 0,67.11 Ao contrário do presente estudo, a IGIV foi administrada na dose de 1 g/kg em dois dias consecutivos e foi considerada resistência se febre persistente após 24 horas de início de terapêutica, ou se recrudescência com sintomas após período apirético. O modelo de Egami foi validado com uma sensibilidade de 0,78 e especificidade de 0,76; no entanto, foi definida resistência se ausência de diminuição do valor de PC-R em mais de 50% e persistência da febre 48 horas após administração de IGIV.12 Loomba et al.25 não conseguiram validar o modelo de Egami, mesmo aplicando-o separadamente à DK clássica, atípica e por etnias. O modelo de Sano, validado com uma sensibilidade de 0,77 e especificidade de 0,86, foi o único dos três a ajustar o tamanho dos AAC à superfície corporal.13 No entanto, também usou administração de IGIV na dose de 1 g/kg em 2 dias consecutivos e definiu resistência se persistência de febre após 24 horas do término da terapêutica. Este trabalho apresenta as limitações inerentes ao fato de ser um estudo retrospectivo e com uma amostra de tamanho reduzido. Conclusão A PC-R e a VS são variáveis independentes que mostraram tendência preditora de resistência à IGIV com pontos de corte ótimos de 15,1 mg/dL e 90,5 mm/h, respectivamente. Existe, no entanto, necessidade de um estudo com uma amostra de dimensões adequadas para validar um modelo baseado nesses dois dados analíticos. As complicações cardíacas não se resumem às artérias coronárias, devendo ser mais abrangentes o estudo e o seguimento desses pacientes. Os modelos validados para a população japonesa apresentam utilidade muito limitada na amostra deste estudo, reforçando ainda mais a necessidade e a importância de novas abordagens. Original Article Kawasaki Disease: Predictors of Resistance to Intravenous Immunoglobulin and Cardiac Complications https://orcid.org/0000-0001-7047-4170 Faim Diogo 1 https://orcid.org/0000-0001-7506-0647 Henriques Cláudio 1 Brett Ana 2 Francisco Andreia 1 Rodrigues Fernanda 2 Pires António 1 1 Coimbra Portugal Centro Hospitalar e Universitário de Coimbra EPE - Cardiologia Pediátrica Coimbra - Portugal 2 Coimbra Portugal Centro Hospitalar e Universitário de Coimbra EPE - Urgência e Unidade de Infeciologia, Coimbra - Portugal Mailing Address: Diogo Faim • Centro Hospitalar e Universitário de Coimbra EPE - Praceta, R. Prof. Mota Pinto, 3075 Coimbra 3000-075 – Portugal. E-mail: diogofaim92@gmail.com Abstract Background: Kawasaki disease (KD) is the leading cause of acquired cardiac disease in children, in developed countries. Objectives: To identify predictive factors for resistance to intravenous immunoglobulin (IVIG), calculate the effectiveness of Japanese predictive models and characterize cardiac complications. Methods: Retrospective analysis of KD cases admitted in a Portuguese paediatric hospital between january 2006 and july 2018. ROC curves were used to determine predictive factors for resistance and the multivariate logistic regression analysis was used to develop the predictive model. A significance level of 5% was used. Results: 48 patients with a median age of 36 months were included. The IVIG resistance was 21%. Echocardiographic anomalies were noted in 46%, with coronary involvement in 25% of the sample population. As predictive variable of resistance, the C-reactive protein (CRP) presented an AUC ROC = 0.789, optimal cut-off value 15.1 mg/dL, sensitivity (Sn) 77.8% and specificity (Sp) 78.9%. The erythrocyte sedimentation rate (ESR) presented an AUC ROC = 0.781, optimal cut-off value 90.5 mm/h, Sn 66.7% and Sp 85.7%. The model with the two variables showed p = 0.042 and AUC ROC = 0.790. Predictive strength of Japanese models were: Kobayashi (Sn 63.6%, Sp 77.3%), Egami (Sn 66.7%, Sp 73.1%), Sano (Sn 28.6%, Sp 94.1%). Conclusion: CRP and ESR are independent variables that were related to IVIG resistance, with optimal cut-off points of 15.1 mg/dL and 90.5 mm/h, respectively. About half of the patients had some form of cardiac involvement. The Japanese models appeared to be inadequate in our population. (Arq Bras Cardiol. 2021; 116(3):485-491) Keywords: Kawasaki Disease/complications Mucocutaneous Lymph Node Syndrome/complications Drug Resistance Coronary Artery Disease Immunoglobulin Child Introduction Kawasaki disease (KD) is an acute self-limiting vasculitis, which affects medium-sized vessels and is the leading cause of acquired cardiac disease in pediatric age groups.1 Its etiology remains uncertain, but several factors have been associated to it, namely genetic, environmental, and inflammatory ones.2 Although with a worldwide distribution, its highest prevalence is in Japan, where the incidence is on the rise.3 In Portugal, an epidemiological study carried out in 2017 showed an mean annual incidence of 6.5 per 100,000 children under 5 years of age.4 Based on the 2004 American Pediatric Academy criteria,5 classic KD is considered if fever persists for five days or more and if at least four of five additional clinical criteria are observed: nonexudative bilateral conjunctivitis, alterations of the lips and oral cavity, erythematous rash, changes in the extremities, and cervical lymphadenopathy. If fever lasts for five or more days and only two or three additional criteria are present, it is considered atypical KD, if supported by laboratory and echocardiographic data.2 If not treated within an established period, KD can be complicated by coronary artery aneurysms (CAA) in up to 25% of cases.2 Although coronary artery involvement is the most feared consequence of the disease, other cardiac complications are possible.2,6–8 Treatment with intravenous immunoglobulin (IVIG) in the acute phase administered in the first 10 days of illness reduces the incidence of CAA to 4%.2 IVIG resistance occurs in 10-20% of cases, increasing the likelihood of coronary involvement.2 There are different possible approaches in case of IVIG resistance, such as a second dose of IVIG, corticosteroids and/or monoclonal antibodies.9 No benefit has been described when corticosteroids are used in addition to IVIG in the first instance and this therapeutics is currently reserved for refractory cases.10 In order to identify the cases that could potentially be resistant to treatment with IVIG, and benefit from adjuvant therapies in the initial phase, models based on a scoring system have been developed. Three have been validated in the Japanese population, namely the Kobayashi,11 Egami,12 and Sano scoring systems.13 However, several studies have shown that these models are poor predictors in many western populations.10,14,15 The aim of this study was to identify clinical and laboratory predictive factors regarding resistance to IVIG and coronary artery involvement, and to develop a more suitable predictor model of resistance in this population. Secondary objectives regard characterizing the KD cases admitted to a central pediatric hospital over a period of 13 years, to verify the effectiveness of the Japanese scoring systems in our population sample and to analyze the non-coronary cardiac complications of KD. Methods Sample Retrospective analysis of KD cases admitted to the Hospital Pediátrico – Centro Hospitalar e Universitário de Coimbra (HP-CHUC) diagnosed from 01/01/2006 to 06/30/2018. All patients between 30 days and <18 years with KD and treated with IVIG at diagnosis at HP-CHUC were included in the study. All the patients transferred from outlying centers with a diagnosis of KD and managed at these institutions were excluded. The diagnosis of typical and atypical KD was based on the American Academy of Pediatrics criteria. We considered day one of fever on the day the fever started, defined as axillar temperature ≥ 38ºC. Resistance to IVIG was considered if the fever persisted 36 hours after its administration. All patients who received corticosteroids simultaneously with the first dose of IVIG were excluded from the resistance quantification. Dallaire z scores were used to classify the coronary artery morphology defining: coronary artery ectasia if z score between 2 and 2.4, small aneurysm if z score between 2.5 and 4.9, medium aneurysm if z score between 5 and 9.9 and absolute dimension < 8 mm, and giant aneurysm if z score ≥ 10 or absolute dimension ≥ 8 mm. If unable to calculate the z score, the absolute dimensions were used, being small aneurysm if ≥ 2.5 mm and < 4mm, medium aneurysm if ≥ 4 mm and < 8 mm and giant aneurysm if ≥ 8 mm. Regarding other cardiac complications, the coronary artery hyperechogenicity and lack of tapering on echocardiography were not considered as echocardiographic diagnostic criteria. To calculate the effectiveness of the Japanese models, all patients who did not have the necessary data to be considered as high or low risk of IVIG resistance were excluded. Scoring and categorization in high or low risk patients were performed as shown in Table 1. Table 1 Scoring system of the Japanese modelss Model Score High risk Kobayashi ≥ 4 points AST > 100 U/L 2 Na ≤ 133 mmol/L 2 IVIG with fever ≤ 4 days 2 Neutrophils/Leucocytes ≥ 80% 2 CRP ≥ 10 mg/dL 1 Age ≤ 1 year old 1 Platelets ≤ 300,000/µL 1 Egami ≥ 3 points ALT ≥ 80 U/L 2 IVIG with fever ≤ 4 days 1 CRP ≥ 8 mg/dL 1 Age ≤ 6 months 1 Platelets ≤ 300,000/µL 1 Sano ≥ 2 points AST > 200 U/L 1 Total bilirubin ≥ 0.9 mg/dL 1 CRP ≥ 7 mg/dL 1 AST: aspartate transaminase; U: international unity; L: liter; Na: serum sodium; mmol: millimole; IVIG: intravenous immunoglobulin; CRP: C-reactive protein; mg: milligram; dL: deciliter; µL: microliter; ALT: alanine transaminase. Statistical Analysis The SPSS® (IBM®, SPSS® Statistics Inc., Chicago) program version 25.0 was used the performed the statistical analysis. The Shapiro-Wilk test was used to test the normality of the variables. The continuous variables with normal distribution were described using mean and standard deviation (SD) and continuous variables without normal distribution were described using median and interquartile range (IQR). We used the Fisher’s exact test to compare categorical variables, the Student’s t-test to compare parametric variables and the Mann-Whitney test to compare the non-parametric ones. The Receiver Operating Characteristic (ROC) curves were used to evaluate the individual discriminative capacity of each variable and to identify the optimal cutoff points to predict resistance to IVIG. The variables were considered as good predictors if the area under the curve (AUC) > 0.75. Multivariate logistic regression analysis was used to develop the predictive resistance model. A significance level of 5% was used. Results Forty-eight patients met the KD criteria, of whom 32 (66.7%) were male. The median age was 36 months (IQR 16.75-89.25), 62.5% of patients were less than five years old and 10.4% over nine years old. On the day of admission, all the patients presented with fever, with a median duration of five days (IQR 4-8), minimum of one day and maximum of 14 days. Among the five main clinical criteria, nonexudative bilateral conjunctivitis was observed in 94% of cases, alterations of the lips and oral cavity in 90%, erythematous rash in 84%, changes in extremities in 75%, and cervical lymphadenopathy in 69%. The most common findings among oral alterations were cheilitis (67%) and lip erythema (67%), followed by erythema of the oropharynx (50%) and the strawberry tongue (48%). The most prevalent changes in the extremities were erythema (52%), followed by swelling (31%) and peeling (25%). Inflammatory signs at the Bacillus Calmette-Guérin (BCG) vaccination site were observed in 23% of patients. Atypical KD was diagnosed in 17% of the cases. The median duration of hospital stay was two days (IQR 1-6.75). During the acute phase, IVIG 2g/kg was administered to all patients. The median day of illness of the administration was 6.5 (IQR 5-8). Simultaneously, 45-100mg/kg of acetylsalicylic acid (ASA) were administered to 47 patients in the acute phase. Five children received corticosteroids together with the first dose of IVIG. After the acute phase, all patients were medicated with ASA 3-5 mg/kg/day, three patients with clopidogrel and one with enoxaparin. Nine patients were resistant to the IVIG (21%), of which one had atypical KD (p = 0.543). All nine repeated IVIG administration, five of which with methylprednisolone 30 mg/kg/day. Among the variables evaluated as predictors of IVIG resistance (Table 2), C-reactive protein (CRP) presented an AUC ROC of 0.78 (95% confidence interval (CI): 0.632 – 0.947), and the erythrocyte sedimentation rate (ESR), an AUC ROC of 0.781 (95%CI: 0.585 – 0.977). The optimal cut-off value for CRP was 15.1 mg/dL with sensitivity (Sn) of 77.8% and specificity (Sp) of 78.9% (Odds ratio (OR) = 13.125 95%CI: 2.271 – 75.858). The optimal cut-off value for ESR was 90.5 mm/h, with Sn of 66.7% and Sp of 85.7% (OR = 12.000 95%CI: 1.718 – 83.803). A logistic model was developed with these two variables, with a p-value of 0.042, AUC ROC of 0.79 (95%CI: 0.589 – 0.992), with Sn of 83.3% and Sp of 77.1%, but with a 25% variance (Nagelkerke R2 = 0.254). Table 2 Receiver operating characteristic analysis of several variables to predict resistance to intravenous hemoglobulin Characteristic AUC [95%CI] Age 0.542 [0.377; 0.708] IVIG administration day 0.595 [0.403; 0.787] Hemoglobin 0.611 [0.416; 0.806] Leucocytes 0.525 [0.331; 0.719] Neutrophils 0.637 [0.447; 0.828] Platelets 0.513 [0.295; 0.732] ESR 0.781 [0.585; 0.977] CRP 0.789 [0.632; 0.947] Na 0.715 [0.475; 0.955] AST 0.648 [0.434; 0.862] ALT 0.693 [0.486; 0.901] Total bilirubin 0.500 [0,139; 0.861] Albumin 0.693 [0.459; 0.928] AUC: area under the curve; CI: confidence interval; IVIG: intravenous immunoglobulin; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; Na: serum sodium; AST: aspartate transaminase; ALT: alanine transaminase. Coronary artery changes were found in 12 children (25%), seven with ectasia and five with CAA. The comparison between groups with and without coronary artery involvement is shown in Table 3. The duration of fever and the use of corticosteroids were the only significant differences between these two groups. Patients with coronary artery involvement had longer duration of fever (p = 0.038) and greater need for corticotherapy (p = 0.009). Four patients had CAA when methylprednisolone was started. Among the five patients with CAA, three met the criteria for small aneurysms, one for medium aneurysms and one for giant ones. These patients are summarized in Table 4. Table 3 Characteristics of groups with and without coronary artery involvement Characteristics Without coronary involvement (n =36) Coronary involvement (n= 12) p - value Corticoid (n=10) n 4 6 0.009 Resistance to IVIG (n=9) n 5 4 0.173 Age (months) Mean ± sd 59.7 ± 57 47.5 ± 30.1 0.35 Fever duration (days) Mean ± sd 7.4 ± 2.8 9.4 ± 3 0.038 IVIG administration day Mean ± sd 6.5 ± 2.9 7.6 ± 3.5 0.283 Atypical KD n 6 2 0.686 Haemoglobin (g/dL) Mean ± sd 11.5 ± 1.3 11.2 ± 1.3 0.359 Leucocytes (/µL) Mean ± sd 14,174 ± 6,010 15,216 ± 6,918 0.619 Neutrophils (/µL) Mean ± sd 9,515 ± 4,770 10,994 ± 5,629 0.378 Platelets (/µL) Mean ± sd 328,389 ± 127,125 362,667 ± 282,652 0.691 CRP (mg/dL) Mean ± sd 10.4 ± 8.8 19.6 ±25 0.243 ESR (mm/h) Mean ± sd 71.6 ± 19 76.7 ± 33.4 0.672 Na (mmol/L) Mean ± sd 137 ± 4 137 ± 5 0.869 AST (U/L) Mean ± sd 64 ± 53 101 ± 91 0.222 ALT (U/L) Mean ± sd 99 ± 116 113 ± 86 0.712 Total bilirubin (mg/dL) Mean ± sd 1.9 ± 2 2.8 ± 3 0.538 Albumin (g/L) Mean ± sd 35.6 ± 4.3 34.9 ± 7.2 0.77 n: absolute value; IVIG: intravenous immunoglobulin; sd: standard deviation; KD: Kawasaki disease; g: gram; dL: deciliter; µL: microliter; CRP: C-reactive protein; mg: milligram; ESR: erythrocyte sedimentation rate; h: hour; Na: serum sodium; mmol: millimole; L: litre; AST: aspartate transaminase; U: international unity; ALT: alanine transaminase. Table 4 Characteristics of patients with coronary artery aneurysms Characteristics Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Age (months) 12 60 108 63 4 Male Yes Yes Yes Yes Yes Fever duration (days) 7 6 8 9 14 Classic KD Yes Yes Yes Yes No Day of fever in IVIG 1st dose 7 6 4 6 14 Resistance to IVIG No No Yes Yes NA Day of fever in IVIG 2nd dose NA NA 6 8 NA MPDN (30mg/Kg/day) With IVIG 1st dose With IVIG 2nd dose No NA NA No NA NA Yes No Yes Yes No Yes Yes Yes No CAA classification Small Small Small Medium Giant Maximum z score NA 4.46 3.56 6.94 13.81 Arteries involved RCA; CT RCA; LCA LCA RCA; LCA RCA; LAD; LCX KD: Kawasaki disease; IVIG: intravenous immunoglobulin; MPDN: methylprednisolone; mg: milligram; Kg: kilogram; CAA: coronary artery aneurysms; RCA: right coronary artery, CT: common trunk; LCA: left coronary artery; LAD: left anterior descending artery; NA: not applicable; LCX: left circumflex artery. In the acute phase, in addition to coronary involvement, 10 patients presented with pericardial effusion, three with mild mitral valve regurgitation, two with left ventricular systolic dysfunction, one with cardiogenic shock and one with variable first degree atrioventricular (AV) block. After the acute phase, the patient with the AV block developed left ventricle (LV) dilation and another patient developed LV hypertrophy. Table 5 summarizes Sn, Sp,and the positive (PPV) and negative predictive values (NPV) for the Japanese models in our sample. Table 5 Statistical values of the Japanese models in our study Model n Sn (%) Sp (%) PPV (%) NPV (%) Kobayasahi 34 63.6 77.3 53.8 81 Egami 39 66.7 73.1 50 82.6 Sano 25 28.6 94.1 66.7 77.3 n: absolute value; Sn: sensitivity; Sp: Specificity; PPV: positive predictive value; NPV: negative predictive value. Discussion Despite the lower incidence compared to Japan, KD is a vasculitis that is still an important cause of pediatric disease in our population. Early diagnosis and management are two important factors that appear to reduce cardiac involvement. Our study revealed an incidence of IVIG resistance similar to the 10 to 20% described in the literature.2 Over the years, efforts have been made to find clinical and laboratory factors that can predict this resistance in order to introduce adjuvant therapies at an early stage of the disease. There are several parameters in the literature that have been studied for this purpose, such as age, serum albumin, transaminases, total bilirubin, neutrophils count, platelet count, CRP, ESR, among others.14,16–20 In our study, CRP and ESR presented a statically significant predictive capacity in relation to IVIG resistance. For CRP, the optimal cut-off point was 15.1 mg/dL (Sn 77.8%, Sp 78.9%, OR 13.125). Patients with CRP values above 15.1 mg/dL are about 13 times more likely to be resistant to IVIG than those with lower values. Concerning ESR, the optimal cut-off point was 90.5 mm/h (Sn 66.7%, Sp 85.7%, OR 12.000). Patients with ESR greater than 90.5 mm/h have a probability of resistance to IVIG approximately 12 times higher than those with lower values. Combining these two independent variables, a statistically significant model was obtained (p = 0.042), whose cut-off point has Sn of 83.3% and Sp of 77.1%. Despite these encouraging results, the variance explained by the model is only 25% (Nagelkerke R2 = 0.254). Thus, although statistically significant, it cannot be validated, which is largely due to the small sample size. Nevertheless, based on these trends, the base-line values for CRP and ESR should be known prior to IVIG administration. The resistance predictor capacity highlights the role of inflammation in this disease, a possible underlying trigger in KD vasculitis.21 The etiology of KD remains uncertain, however, predisposing factors have been put forward. One, is the immaturity of the immune system, a theory that is supported by the fact that KD predominantly affects children under the age of five. In our study, 62.5% of the patients belonged to this age group, which, although corresponding to the majority of the sample, is below the 80% described in the literature.1 A possible explanation for this result is genetic contribution, since the incidences described in the literature are from studies with a wide range of ethnicities, including Asian children. Coronary artery involvement occurred in 12 (25%) children, seven with ectasia and five with CAA. Therefore, the incidence of CAA was 10%, which is higher than the 4% reported in the literature. Comparing the groups with and without coronary artery involvement, a statistically significant difference was found regarding the duration of fever (p = 0.038). This result highlights the deleterious effects of persistent fever and the need for IVIG administration, preferably up to the tenth day of the disease, in order to avoid cardiac sequelae.9 The use of corticosteroids in KD is still a topic of debate and controversy. The most consensual is the use intravenous methylprednisolone (MPDN) at a dose of 15 to 30 mg/kg/day, for three days.9 In patients with refractory KD, MPDN supresses the inflammatory cytokine levels more quickly than a second dose of IVIG,9 although it is not recommended as a first-line treatment. Sleeper et al.10 evaluated the impact of corticosteroids at different times of the disease and showed that, with regards to the development of CAA, the only statistically significant difference was in those refractory to the first dose of IVIG which combined corticosteroids with the second dose of IVIG. The cardiac complications and echocardiographic findings, others than coronary artery involvement were also evaluated. Three cases of left ventricular systolic dysfunction were identified, one of which with cardiogenic shock, a complication also described in the literature.2,7 In the acute phase, ten patients presented with pericardial effusion without hemodynamic compromise, three patients with mild mitral valve regurgitation and one with first degree AV block. Chbeir et al.22 found a relation between resistance to IVIG, CAA, and initial cardiac echocardiographic findings such as pericardial effusion, coronary hyperechogenicity, and coronary ectasia. The coronary hyperechogenicity and the lack of tapering on echocardiography were not considered as relevant factors, since they are subjective findings, poorly reproducible and can be found both in febrile illnesses and in healthy children.23 During the chronic phase, one patient remained with conduction system impairment and developed LV dilation, and another patient developed LV hypertrophy. The long-term cardiac repercussions in KD remain unclear. Friedman et al.6 reported an increase in the occurrence of long-term adverse cardiac effects, leading to primary angioplasty, coronary bypass surgery, heart transplantation, and death, in patients who developed CAA with higher z scores and who were initially resistant to IVIG. A study by Holve et al.8 revealed a low incidence of adverse cardiac effects in subjects up to 21 years of age, but a greater likelihood of developing high blood pressure from the age of 15. Japanese predictive models presented poor clinical utility in this study (Table 4). The model with the highest specificity was Sano’s, although with very low sensitivity and with only a small number of cases included. Egami’s model was the most sensitive, however, not powerful enough to be validated. The genetic component may be the explanation for these differences. In fact, the results presented here are similar to those of other studies carried out outside Japan, in which none were able to validate the models in their samples.10,14–17,20 It is still important to note the differences in the study design in relation to the Japanese models, namely that they were applied only to patients with classical KD. Another Japanese study failed to validate the models in a sample exclusively composed of atypical KD cases.24 The Kobayashi model was validated for the Japanese population with Sn of 86% and Sp of 67%.11 Contrary to the present study, IVIG was administered at a dose of 1 g/kg on two consecutive days and resistance was considered if fever persisted 24 hours after the beginning of the treatment, or in case of recurrence after a period without fever. The Egami model was validated with Sn of 78% and Sp of 76%, however, resistance was considered if the CRP value did not decrease by more than 50% and fever persisted for longer than 48 hours after IVIG administration.12 Loomba et al.25 were not able to validate the Egami model even when applying it separately to classical and atypical KD, and by ethnicity. The Sano model, validated with Sn of 77% and Sp of 86%, was the only one of the three to adjust the size of the CAA to the body surface.13 However, it also used IVIG at a dose of 1 g/kg on two consecutive days and defined resistance if fever persisted 24 hours after the end of therapy. The main limitations of this analysis are related to its retrospective methodology and sample size. Conclusions CRP and ESR are independent variables that showed a predictive trend regarding resistance to IVIG, with optimal cut-off values of 15.1 mg/dL and 90.5 mm/h, respectively. However, there is a need for a multicenter study with a sample of adequate dimensions to validate a model based on these two analytical parameters. Cardiac complications are not limited to coronary arteries, and the study and follow-up of these patients should be more widespread. The validated models for the Japanese population have very limited utility in our population, further reinforcing the need and importance of new approaches. Fontes de Financiamento O presente estudo não contou com fontes de financiamento externas. Vinculação Acadêmica Não há vinculação deste estudo a programas de pós-graduação. Aprovação Ética e Consentimento Informado Este artigo não contém estudos com humanos ou animais realizados por nenhum dos autores. Author Contributions Conception and design of the research: Faim D, Henriques C, Brett A, Francisco A, Rodrigues F, Pires A; Data acquisition: Faim D, Henriques C; Analysis and interpretation of the data: Faim D, Brett A, Francisco A; Statistical analysis and Writing of the manuscript: Faim D; Critical revision of the manuscript for intellectual content: Faim D, Brett A, Francisco A, Rodrigues F, Pires A. Potential Conflict of Interest The authors report no conflict of interest concerning the materials and methods used in this study or the findings specified in this paper. Sources of Funding There was no external funding source for this study. Study Association This study is not associated with any thesis or dissertation. Ethics Approval and Consent to Participate This article does not contain any studies with human participants or animals performed by any of the authors.	30 MILLIGRAM/KILOGRAM, QD	DrugDosageText	CC BY	33470332	19,012,805	2021-03
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug reaction with eosinophilia and systemic symptoms'.	Dress syndrome in 7-year-old male child - case report. Drug rash with eosinophilia and systemic symptoms (DRESS) is a severe drug-induced hypersensitivity reaction, which, due to the asymptomatic beginning and non-specific nature of symptoms, is hard to identify. This report presents the case of a 7-year-old boy, who was referred to the Department of Paediatric Surgery with fever up to 38°C, vomiting and diarrhoea, accompanied by erythematous, maculopapular rash. Based on laboratory and radiology tests and specific diagnostic criteria, DRESS syndrome was diagnosed. The presented case report emphasises the need to carry out differential diagnosis, including the potentially life-threatening DRESS syndrome, with common symptoms in children such as fever and rash. pmcIntroduction Drug rash with eosinophilia and systemic symptoms (DRESS) syndrome encompasses drug rash with eosinophilia and organ impairment symptoms. It was first described in 1996 by Bocquet et al. (1). The name was coined by Callot et al. (2) in a study of 21 patients with acute organ disease, eosinophilia and skin symptoms. The frequency of DRESS is estimated to be between 1/1,000 and 1/10,000 patients, and although this frequency may well be higher, it is rarely diagnosed (3). The pharmacological compounds commonly associated with DRESS syndrome are phenytoin and phenobarbital (4). The clinical symptoms of DRESS syndrome appear 2–8 weeks after taking the reaction-inducing drug; they typically manifest as fever, maculopapular rash, peripheral lymphadenopathy, haematological disorders and impairment of one or more organs (3). Mortality is estimated to be 10% in adults (4) and 5.4% in children (5). Case report A 7-year-old boy, M.C., was referred to the Department of Pediatrics, Allergology and Gastroenterology in Bydgoszcz with a rash and fever. On interviewing, we found that he had attended the Department of Paediatric Surgery 3 weeks previously for the treatment of appendicitis complicated by peritonitis. One week after appendectomy, he required re-laparotomy for bowel obstruction. Intensive parenteral antibiotic therapy was applied: cefotaxime, metronidazole and amikacin initially, followed by vancomycin, imipenem with cilastatin and fluconazole. After clinical improvement and normalisation of the inflammatory markers, he was discharged in good condition. The next day, the boy experienced vomiting and diarrhoea (up to six loose stools per day). Three days later, he experienced a 38°C fever accompanied by erythematous rash covering the earlobes, face and torso. Therefore, the next day, the boy was readmitted to hospital for further diagnosis and treatment. Upon admission, the patient was assessed to be in good general condition. Apart from the surgical scar in the abdominal area, provided with stitches, physical examination revealed an intense red, maculopapular rash throughout the body, being most severe on the face. Laboratory tests revealed eosinophilia (21%) and elevated C-reactive protein (CRP; 67 mg/L). Initially, rupatadine was administered. In the following days, fever up to 39°C, diarrhoea and rash with a tendency to merge were observed. Additionally, the boy developed swelling of the subcutaneous tissue, with the most intense skin lesions being observed on the cheeks, earlobes and lips. The following day, an increase in both inflammatory parameters and eosinophilia was observed. The results of all the laboratory tests are presented in Table 1. Table 1 The results of laboratory tests during hospitalisation Results of the tests Laboratory test Laboratory norm Day of hospitalisation 1 3 5 7 16 CRP (mg/L) <5.0 67.5 126.45 47.44 0.83 Procalcitonin (ng/mL) <0.5 0.81 8.09 0.4 Eosinophilia (%) 2–4 21 30 36 0.56 Haemoglobin (g/dL) 12.0–15.0 11.1 10.1 8.6 9.7 APTT (sec) 27–40 47.2 53.5 32.5 D-Dimers (ng/mL) <500 1510 2578 900 Albumin (g/dL) 3.8–5.4 4.3 3.0 4.5 Protein in the urine (mg/dL) Not present 10 50 Not present Creatinine (mg/dL) 0.3–0.7 1.13 1.08 0.54 ALT (U/L) <44 12 12 12 AST (U/L) <48 18 12 14 TSH (mIU/mL) 0.3500– 4.9400 5.9817 fT4 (ng/dL) 0.70–1.48 1.26 fT3 (pg/mL) 1.71–3.71 2.18 CRP, C-reactive protein; APTT, activated partial thromboplastin time; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TSH, thyroid-stimulating hormone; fT3, free tri-iodothyronine; fT4, free thyroxine. Initially, the patient was treated with cefuroxime and antipyretic drugs. The rash had a tendency to periodically merge, showing the target shield symptom (Figure 1). DRESS Syndrome was suspected. No infection with viruses, including hepatitis A, B and C viruses (HAV, HBV and HCV), human immunodeficiency virus (HIV), cytomegalovirus (CMV) or Epstein–Barr virus (EBV), was confirmed. Abdominal ultrasonography revealed slight enlargement of the liver, spleen and kidneys and the presence of fluid in the peritoneum. The abdominal circumference increased over the following days. Cardiac echocardiography identified fluid in the pericardial sac. Figure 1 Target shield symptom. Figure 2 Massive maculopapular lesions covering the skin of the entire body. Based on the overall clinical picture and the current criteria, DRESS syndrome was diagnosed. On Day 7, methylprednisolone was injected intravenously for 8 days. The patient’s ionic disturbances were equalised, and a single albumin transfusion was administered. Due to the presence of pericardial fluid, spironolactone was given as recommended by the consulting cardiologist. Over the subsequent days, clinical improvement was observed: gradual regression of rash and fever, normalisation of inflammation markers and haemostasis parameters, reduction in the sizes of the liver and spleen and reduction in the amount of fluid in the pericardium and peritoneal sac. The boy was discharged from hospital in a good general condition with the recommendation to continue taking oral prednisone in reducing doses and spironolactone for the next 7 days. Written informed consent was obtained from the patient and his mother for the publication of this case report. Discussion DRESS is a systemic, severe, drug hypersensitivity syndrome generally observed in adults (3), but it is rarely seen in children (5). Therefore, the presented case is a valuable supplement to the available literature. DRESS is a drug-induced reaction (3) manifesting as a very wide spectrum of clinical symptoms, including fever >38°C; maculopapular rash, pruritus, erythroderma and exfoliation, occurring mainly on the skin of the face, upper torso and on the extremities, with a tendency to merge within the most intense skin lesions; painful peripheral lymphadenopathy, abdominal pain and involvement of internal organs – liver, kidneys, lungs and heart; and changes in peripheral blood profile (eosinophilia, neutrophilia, neutropaenia, thrombocytopaenia, haemolytic anaemia and presence of atypical lymphocytes in peripheral blood) (3, 5). Diagnosis is often complicated by the long asymptomatic period between taking the drug and the appearance of symptoms, in addition to the non-specific nature of the symptoms. The most frequently presented symptoms, i.e. fever and rash, are indicative of many childhood diseases, including sepsis. A more specific symptom of DRESS is eosinophilia; however, this is observed in only 30% of patients (6). Due to this variability of the clinical picture, a European criterion for recognising severe cutaneous adverse reactions and drug responses (namely, the Registry of Severe Cutaneous Adverse Reactions or RegiSCAR) has been prepared to facilitate rapid diagnosis (Table 2). (7) Table 2 The RegiSCAR-Group Diagnosis Score for drug rash with eosinophilia and systemic symptoms (DRESS) (12) Symptom No Yes Unknown Fever (≥ 38.5°C) −1 0 −1 Enlarged lymph nodes (>=sites, >=1 cm) 0 1 0 Atypical lymphocytes 0 1 0 Eosinophilia, cells/μL 0 0 700–1,499 or 10%–19.9% 1 ≥ 1,500 or ≥20% 2 Skin rash 0 0 Extent: >50% 0 1 0 At least two of these: oedema, infiltration, purpura, scaling −1 1 0 Biopsy suggesting DRESS −1 0 0 Internal organ involved 0 0 One 1 Two or more 2 Resolution in >=15 days −1 0 −1 At least three biological investigations performed and negative to exclude alternative diagnosis 0 1 0 Final score: >=2 indicates no case; 2–3 indicates possible case; 4–5 indicates probable case; >=5 indicates definite case The presented patient was awarded seven points according to RegiSCAR, indicating the following: eosinophilia of peripheral blood >=20% (2 points), erythematous rash with accompanying oedema of the lips and ears (2 points), involvement of internal organs (fluid in the pericardial and peritoneal sac) (2 points) and timing (occurrence of the first symptoms 2 weeks after the end of antibiotic therapy), as well as the exclusion of another, alternative diagnosis (1 point). Although the pathogenesis of the disease is not fully understood, it may be associated with the accumulation of drug metabolites, caused by an innate deficiency of detoxifying enzymes (3). Human leucocyte antigens (HLA) may also play a role, e.g. the HLA-B1502 phenotype is associated with hypersensitivity to carbamazepine, and the HLA-B1508 phenotype with hypersensitivity to allopurinol (8). A primary or reactivated viral infection may also be involved (EBV, human herpes viruses 6 and 7 [HHV-6, HHV-7]) (3). Of course, different mechanisms may overlap at the molecular level and complex interactions may be possible. DRESS may also be associated with the presence of biologically active metabolites of drugs in the blood due to cytochrome dysfunction. Impaired detoxification of these metabolites causes the activation of keratinocytes, macrophages, eosinophils and T-lymphocytes. Upon binding to tissue compatibility antigens on keratinocytes, active drug metabolites stimulate lymphocyte activity, intensify interleukin (IL)-4 and IL-5 production and increase the number of cytotoxic lymphocytes (8, 9). DRESS syndrome is most often caused by anticonvulsants (carbamazepine, phenytoin, lamotrigine and phenobarbital), anti-tuberculous drugs, allopurinol, sulphasalazine, antibiotics (sulphonamides, vancomycin) and ibuprofen (7). In our patient, an important factor provoking the development of DRESS syndrome appeared to be vancomycin taken during the initial hospitalisation. Symptoms occurred 2 weeks after the end of vancomycin therapy, which is consistent with the diagnostic criteria of DRESS. Differentiation of DRESS includes infection with (EBV), (CMV), hepatitis A, B and C viruses (HAV, HBV, HCV); tuberculosis, toxoplasmosis, borreliosis, lambliasis, parvovirus B19 infection, Kawasaki disease, autoimmune diseases, lymphoproliferative diseases, vasculitis, collagenosis, adult Still disease, as well as neoplastic diseases (3). This differential diagnosis was also carried out in our patient. Treatment consists of discontinuation of the drug that causes the hypersensitivity reaction and implementation of systemic steroid therapy (10). In severe forms resistant to standard treatment, high-dose steroidotherapy (30 mg/kg/day), systemic steroid pulses, intravenous immunoglobulin therapy or plasmapheresis is used (10). The described case is an example of a smooth course of DRESS: the use of steroid therapy in standard doses resulted in the disappearance of symptoms. Conclusion DRESS syndrome is a severe drug-induced hypersensitivity reaction. Diagnosis is complicated by the non-specificity of the clinical picture and the initial asymptomatic period. DRESS should be considered in the diferential diagnosis of children presenting with fever and rash following anticonvulsant or antibiotic treatment. It is extremely important to diagnose this potentially fatal syndrome quickly and implement appropriate treatment. Abbreviations DRESS drug rash with eosinophilia and systemic symptoms; HLA human leucocyte antigens. Conflicts of Interest The authors have no conflicts of interest relevant to this article to disclose. Financial disclosures The authors have no financial relationships relevant to this article to disclose. Consent for publication All authors agree to the publication. Patient’s consent Patient and his mother agree for the publication of this case report.	AMIKACIN, CEFOTAXIME SODIUM, CILASTATIN, FLUCONAZOLE, IMIPENEM, METRONIDAZOLE, VANCOMYCIN	DrugsGivenReaction	CC BY	33470957	20,399,957	2021-01-12
What was the outcome of reaction 'Drug reaction with eosinophilia and systemic symptoms'?	Dress syndrome in 7-year-old male child - case report. Drug rash with eosinophilia and systemic symptoms (DRESS) is a severe drug-induced hypersensitivity reaction, which, due to the asymptomatic beginning and non-specific nature of symptoms, is hard to identify. This report presents the case of a 7-year-old boy, who was referred to the Department of Paediatric Surgery with fever up to 38°C, vomiting and diarrhoea, accompanied by erythematous, maculopapular rash. Based on laboratory and radiology tests and specific diagnostic criteria, DRESS syndrome was diagnosed. The presented case report emphasises the need to carry out differential diagnosis, including the potentially life-threatening DRESS syndrome, with common symptoms in children such as fever and rash. pmcIntroduction Drug rash with eosinophilia and systemic symptoms (DRESS) syndrome encompasses drug rash with eosinophilia and organ impairment symptoms. It was first described in 1996 by Bocquet et al. (1). The name was coined by Callot et al. (2) in a study of 21 patients with acute organ disease, eosinophilia and skin symptoms. The frequency of DRESS is estimated to be between 1/1,000 and 1/10,000 patients, and although this frequency may well be higher, it is rarely diagnosed (3). The pharmacological compounds commonly associated with DRESS syndrome are phenytoin and phenobarbital (4). The clinical symptoms of DRESS syndrome appear 2–8 weeks after taking the reaction-inducing drug; they typically manifest as fever, maculopapular rash, peripheral lymphadenopathy, haematological disorders and impairment of one or more organs (3). Mortality is estimated to be 10% in adults (4) and 5.4% in children (5). Case report A 7-year-old boy, M.C., was referred to the Department of Pediatrics, Allergology and Gastroenterology in Bydgoszcz with a rash and fever. On interviewing, we found that he had attended the Department of Paediatric Surgery 3 weeks previously for the treatment of appendicitis complicated by peritonitis. One week after appendectomy, he required re-laparotomy for bowel obstruction. Intensive parenteral antibiotic therapy was applied: cefotaxime, metronidazole and amikacin initially, followed by vancomycin, imipenem with cilastatin and fluconazole. After clinical improvement and normalisation of the inflammatory markers, he was discharged in good condition. The next day, the boy experienced vomiting and diarrhoea (up to six loose stools per day). Three days later, he experienced a 38°C fever accompanied by erythematous rash covering the earlobes, face and torso. Therefore, the next day, the boy was readmitted to hospital for further diagnosis and treatment. Upon admission, the patient was assessed to be in good general condition. Apart from the surgical scar in the abdominal area, provided with stitches, physical examination revealed an intense red, maculopapular rash throughout the body, being most severe on the face. Laboratory tests revealed eosinophilia (21%) and elevated C-reactive protein (CRP; 67 mg/L). Initially, rupatadine was administered. In the following days, fever up to 39°C, diarrhoea and rash with a tendency to merge were observed. Additionally, the boy developed swelling of the subcutaneous tissue, with the most intense skin lesions being observed on the cheeks, earlobes and lips. The following day, an increase in both inflammatory parameters and eosinophilia was observed. The results of all the laboratory tests are presented in Table 1. Table 1 The results of laboratory tests during hospitalisation Results of the tests Laboratory test Laboratory norm Day of hospitalisation 1 3 5 7 16 CRP (mg/L) <5.0 67.5 126.45 47.44 0.83 Procalcitonin (ng/mL) <0.5 0.81 8.09 0.4 Eosinophilia (%) 2–4 21 30 36 0.56 Haemoglobin (g/dL) 12.0–15.0 11.1 10.1 8.6 9.7 APTT (sec) 27–40 47.2 53.5 32.5 D-Dimers (ng/mL) <500 1510 2578 900 Albumin (g/dL) 3.8–5.4 4.3 3.0 4.5 Protein in the urine (mg/dL) Not present 10 50 Not present Creatinine (mg/dL) 0.3–0.7 1.13 1.08 0.54 ALT (U/L) <44 12 12 12 AST (U/L) <48 18 12 14 TSH (mIU/mL) 0.3500– 4.9400 5.9817 fT4 (ng/dL) 0.70–1.48 1.26 fT3 (pg/mL) 1.71–3.71 2.18 CRP, C-reactive protein; APTT, activated partial thromboplastin time; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TSH, thyroid-stimulating hormone; fT3, free tri-iodothyronine; fT4, free thyroxine. Initially, the patient was treated with cefuroxime and antipyretic drugs. The rash had a tendency to periodically merge, showing the target shield symptom (Figure 1). DRESS Syndrome was suspected. No infection with viruses, including hepatitis A, B and C viruses (HAV, HBV and HCV), human immunodeficiency virus (HIV), cytomegalovirus (CMV) or Epstein–Barr virus (EBV), was confirmed. Abdominal ultrasonography revealed slight enlargement of the liver, spleen and kidneys and the presence of fluid in the peritoneum. The abdominal circumference increased over the following days. Cardiac echocardiography identified fluid in the pericardial sac. Figure 1 Target shield symptom. Figure 2 Massive maculopapular lesions covering the skin of the entire body. Based on the overall clinical picture and the current criteria, DRESS syndrome was diagnosed. On Day 7, methylprednisolone was injected intravenously for 8 days. The patient’s ionic disturbances were equalised, and a single albumin transfusion was administered. Due to the presence of pericardial fluid, spironolactone was given as recommended by the consulting cardiologist. Over the subsequent days, clinical improvement was observed: gradual regression of rash and fever, normalisation of inflammation markers and haemostasis parameters, reduction in the sizes of the liver and spleen and reduction in the amount of fluid in the pericardium and peritoneal sac. The boy was discharged from hospital in a good general condition with the recommendation to continue taking oral prednisone in reducing doses and spironolactone for the next 7 days. Written informed consent was obtained from the patient and his mother for the publication of this case report. Discussion DRESS is a systemic, severe, drug hypersensitivity syndrome generally observed in adults (3), but it is rarely seen in children (5). Therefore, the presented case is a valuable supplement to the available literature. DRESS is a drug-induced reaction (3) manifesting as a very wide spectrum of clinical symptoms, including fever >38°C; maculopapular rash, pruritus, erythroderma and exfoliation, occurring mainly on the skin of the face, upper torso and on the extremities, with a tendency to merge within the most intense skin lesions; painful peripheral lymphadenopathy, abdominal pain and involvement of internal organs – liver, kidneys, lungs and heart; and changes in peripheral blood profile (eosinophilia, neutrophilia, neutropaenia, thrombocytopaenia, haemolytic anaemia and presence of atypical lymphocytes in peripheral blood) (3, 5). Diagnosis is often complicated by the long asymptomatic period between taking the drug and the appearance of symptoms, in addition to the non-specific nature of the symptoms. The most frequently presented symptoms, i.e. fever and rash, are indicative of many childhood diseases, including sepsis. A more specific symptom of DRESS is eosinophilia; however, this is observed in only 30% of patients (6). Due to this variability of the clinical picture, a European criterion for recognising severe cutaneous adverse reactions and drug responses (namely, the Registry of Severe Cutaneous Adverse Reactions or RegiSCAR) has been prepared to facilitate rapid diagnosis (Table 2). (7) Table 2 The RegiSCAR-Group Diagnosis Score for drug rash with eosinophilia and systemic symptoms (DRESS) (12) Symptom No Yes Unknown Fever (≥ 38.5°C) −1 0 −1 Enlarged lymph nodes (>=sites, >=1 cm) 0 1 0 Atypical lymphocytes 0 1 0 Eosinophilia, cells/μL 0 0 700–1,499 or 10%–19.9% 1 ≥ 1,500 or ≥20% 2 Skin rash 0 0 Extent: >50% 0 1 0 At least two of these: oedema, infiltration, purpura, scaling −1 1 0 Biopsy suggesting DRESS −1 0 0 Internal organ involved 0 0 One 1 Two or more 2 Resolution in >=15 days −1 0 −1 At least three biological investigations performed and negative to exclude alternative diagnosis 0 1 0 Final score: >=2 indicates no case; 2–3 indicates possible case; 4–5 indicates probable case; >=5 indicates definite case The presented patient was awarded seven points according to RegiSCAR, indicating the following: eosinophilia of peripheral blood >=20% (2 points), erythematous rash with accompanying oedema of the lips and ears (2 points), involvement of internal organs (fluid in the pericardial and peritoneal sac) (2 points) and timing (occurrence of the first symptoms 2 weeks after the end of antibiotic therapy), as well as the exclusion of another, alternative diagnosis (1 point). Although the pathogenesis of the disease is not fully understood, it may be associated with the accumulation of drug metabolites, caused by an innate deficiency of detoxifying enzymes (3). Human leucocyte antigens (HLA) may also play a role, e.g. the HLA-B1502 phenotype is associated with hypersensitivity to carbamazepine, and the HLA-B1508 phenotype with hypersensitivity to allopurinol (8). A primary or reactivated viral infection may also be involved (EBV, human herpes viruses 6 and 7 [HHV-6, HHV-7]) (3). Of course, different mechanisms may overlap at the molecular level and complex interactions may be possible. DRESS may also be associated with the presence of biologically active metabolites of drugs in the blood due to cytochrome dysfunction. Impaired detoxification of these metabolites causes the activation of keratinocytes, macrophages, eosinophils and T-lymphocytes. Upon binding to tissue compatibility antigens on keratinocytes, active drug metabolites stimulate lymphocyte activity, intensify interleukin (IL)-4 and IL-5 production and increase the number of cytotoxic lymphocytes (8, 9). DRESS syndrome is most often caused by anticonvulsants (carbamazepine, phenytoin, lamotrigine and phenobarbital), anti-tuberculous drugs, allopurinol, sulphasalazine, antibiotics (sulphonamides, vancomycin) and ibuprofen (7). In our patient, an important factor provoking the development of DRESS syndrome appeared to be vancomycin taken during the initial hospitalisation. Symptoms occurred 2 weeks after the end of vancomycin therapy, which is consistent with the diagnostic criteria of DRESS. Differentiation of DRESS includes infection with (EBV), (CMV), hepatitis A, B and C viruses (HAV, HBV, HCV); tuberculosis, toxoplasmosis, borreliosis, lambliasis, parvovirus B19 infection, Kawasaki disease, autoimmune diseases, lymphoproliferative diseases, vasculitis, collagenosis, adult Still disease, as well as neoplastic diseases (3). This differential diagnosis was also carried out in our patient. Treatment consists of discontinuation of the drug that causes the hypersensitivity reaction and implementation of systemic steroid therapy (10). In severe forms resistant to standard treatment, high-dose steroidotherapy (30 mg/kg/day), systemic steroid pulses, intravenous immunoglobulin therapy or plasmapheresis is used (10). The described case is an example of a smooth course of DRESS: the use of steroid therapy in standard doses resulted in the disappearance of symptoms. Conclusion DRESS syndrome is a severe drug-induced hypersensitivity reaction. Diagnosis is complicated by the non-specificity of the clinical picture and the initial asymptomatic period. DRESS should be considered in the diferential diagnosis of children presenting with fever and rash following anticonvulsant or antibiotic treatment. It is extremely important to diagnose this potentially fatal syndrome quickly and implement appropriate treatment. Abbreviations DRESS drug rash with eosinophilia and systemic symptoms; HLA human leucocyte antigens. Conflicts of Interest The authors have no conflicts of interest relevant to this article to disclose. Financial disclosures The authors have no financial relationships relevant to this article to disclose. Consent for publication All authors agree to the publication. Patient’s consent Patient and his mother agree for the publication of this case report.	Recovered	ReactionOutcome	CC BY	33470957	20,399,957	2021-01-12
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug ineffective'.	A case study of the first pregnant woman with COVID-19 in Bukavu, eastern Democratic Republic of the Congo. BACKGROUND Vertical transmission of covid-19 is possible; its risk factors are worth researching. The placental changes found in pregnant women have a definite impact on the foetus. METHODS We report a case of a 25-year-old woman, gravida 3, para 2 (2 alive children), with a history of two caesarean deliveries, who was infected by the SARS-CoV-2 during the last term of her pregnancy. She gave birth by caesarean at 34 weeks of gestation to a newborn baby also infected with SARS-CoV-2. The peri-operative observations noted several eruptive lesions in the pelvis, bleeding on contact. Microscopic examination of the foetal appendages revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels. CONCLUSIONS This case is one of the first documented cases of COVID-19 in pregnancy in sub-Saharan Africa. We strongly suggest obstetricians to carefully examine the aspect of the peritoneum, viscera and foetal appendages in affected pregnant women. Background In December 2019, first cases of severe acute respiratory syndrome (SARS) due to a new coronavirus (SARS-CoV-2) were reported from Wuhan in China. Soon after, the disease, subsequently named “the 2019 novel coronavirus disease” (COVID-19) and declared a pandemic by the World Health Organisation (WHO) [1], has resulted in over 18.9 million confirmed cases and more than 709,000 deaths worldwide [2]. The pregnant woman can be considered to be more at risk of severe form than the non pregnant woman [3]. Their fragile immunity and frequent comorbidities such as obesity, diabetes mellitus, arterial hypertension, or cardiovascular diseases may expose them at higher risks of developing severe forms of the disease [4] and to adverse pregnancy outcomes, especially during the third trimester [5]. COVID-19 causes pneumonia with acute respiratory distress syndrome (ARDS), which can compromise natural delivery, increase maternal morbidity, or even lead to maternal death [6]. Knowledge about coronavirus disease during pregnancy is still limited [4], and vertical transmission in utero is not yet well established [4, 5]. The risk of mother-to-child transmission of COVID-19 seems to be low [7–9]. Cases of perinatal transmission of COVID-19 have been described, but it is still unclear if this occurred via the transplacental or other routes during delivery [10]. Furthermore, COVID-19 may constitute a threat of premature delivery, intrauterine growth retardation, premature rupture of the membranes, in-utero foetal death or even a premature neonatal death during delivery or soon after [8]. Among pregnant women with SARS-CoV-2, preterm birth is reported 12.9% [11]. There are no established guidelines about the best timing nor the best mode of delivery in COVID-19 infected pregnant women to optimize foetal and maternal well-being [12]. A study on the association between mode of delivery and maternal and neonatal outcomes in COVID-19 patients in Spain has shown that caesarean section was associated with an increased risk for maternal clinical deterioration which remained significant after adjustment for confounders [13]. However, a few case-reports have shown a benefit of a caesarean section on the improvement of respiratory distress in severely affected patients [5, 12]. There have been only a few studies that have investigated the intraoperative findings in women undergoing caesarean delivery [14], or changes in the foetal appendages that may explain the risk of maternal-foetal transmission [15]. We report the first documented case of COVID-19 in a pregnant woman recorded in the province of South Kivu, in eastern Democratic Republic of the Congo (DRC) who gave birth by cesarean section to a premature newborn also infected by SARS-CoV-2. Her pelvic organs exhibited a particular inflammatory appearance, and fetal appendages revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels. Case presentation A 25-year-old woman, gravida 3 para 2, at 34 weeks of gestation, with no medical history of cardiovascular nor other chronic diseases, was admitted to the labour and delivery unit of the “Hôpital Provincial Général de Référence de Bukavu” (HPGRB), in South-Kivu, for preterm labour contractions in a context of COVID-19. She had an history of 2 previous caesarean sections (the first one due to a cervical dystocia and the second indicated because of the prior caesarean), her last born wasaged 16 months. Her husband, tested negative for SARS-CoV-2, was a contact person of a COVID-19 confirmed case. Three weeks before admission, she complained of fever, not responding to acetaminophen. Her obstetrician prescribed her antibiotics, anti-malaria, and anti-spasmodic drugs. Two weeks later, as fever persisted despite all these medications, a reverse transcriptase-polymerase chain reaction (RT-PCR) nasopharyngeal swabs was performed and confirmed she was infected by SARS-CoV-2. She was then admitted to the provincial COVID-19 treatment center for isolation and health care. Upon arrival to the center, her body temperature was 38.7 °C. Gynecologic examination was unremarkable. All bacteriological tests, including hemocultures and cultures of urines were negative. She received antipyretics (acetaminophen), antispasmodics trimethylphloroglucinol and antibiotics (oral azithromycin for 5 days and intravenous ceftriaxone). Two days later, she complained of hypogastric pain, like uterine contractions of low intensity. Obstetricians of the HPGRB were contacted and recommended the administration of antispasmodics intravenously in perfusion. Despite this treatments, fever and uterine contractions persisted, so intravenous dexamethasone 12 mg daily was administered for fetal pulmonary maturation, associated with a tocolysis using nifedipine for 48 h. As the frequency, intensity and duration of contractions increased, accompanied by cervical changes (dilation, effacement, softening, and movement to a more anterior position), the patient was transferred to the labour and delivery unit of the HPGRB for an optimal care. A rapid SARS-Cov-2 antigen test was performed and found to be negative. On admission at the HPGRB, the patient had a good general condition. Her temperature (36.5 °C) and blood pressure (120/60 mmHg) were normal. The uterine height was 29 cm, the fœtus was in cephalic presentation. On vaginal examination, the uterine cervix was softened, median, 5 mm long and had a 5 cm dilatation. Membranes were intact and the fœtal head was mobile. An obstetrical ultrasound confirmed the cephalic presentation and estimated the foetal weight at 1600 g. Foetal monitoring confirmed a foetal well-being, with a stable foetal cardiac rhythm around 140 beats per minute. Tocography showed two to three contractions per minute and an intensity of 50 to 60 mmHg. A diagnosis of intractable preterm labor in a COVID-19 patient with a history of iterative caesarean deliveries was made. A classic Caesarean section with a Pfannestiel incision was performed. The peritoneal cavity and uterus were found to be very inflamed. Fetal appendages as well as the bladder were strewn with eruptive, vesicular lesions bleeding on contact (Figs. 1 and 2). The amniotic fluid was opalescent. The placenta weighed 500 g and had a clot on the maternal side on less than 20% of the surface. Anatomopathological examination subsequently revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels (Figs. 3 and 4), and a diffuse hyalinization with marked angiogenesis of the villous stroma. Fig. 1 Peri-operative appearance showing several bleeding eruptive lesions on contact ( ) with the posterior surface of the uterus, on the ovaries, the left fallopian tube up to Douglas’ pouch Fig. 2 Same lesions in the right appendages of the uterus ( ) Fig. 3 Section of the placenta; in the Hematoxylin-eosin (HE) staining we noted on sections of the placenta at 100X magnification, highly vascularized villi, with vascular lesions ( ) in the form of congestion and thrombosis visualized in certain fields and large areas of stromal hyalinization Fig. 4 Cross section of the umbilical cord (Magnification: 40X, Hematoxylline-eosin staining). There is the presence of thrombus ( ) in the lumen of the umbilical vessel, Wharton’s jelly is without distinction, no inflammatory reaction About five minutes after skin incision, a female newborn weighing 1760 g was delivered with 1 and 5 min APGAR scores of 9–10. The newborn was immediately transferred to the neonatal ward for specialized neonatal treatment for an optimal care and to minimise the potential risk of infection. Gestational age was estimated at 33 weeks according to the Finnstrom score. The newborn received the usual care (drying, stimulation, vitamin K1, Fluorescein and care of the umbilical cord). A gastric liquid was collected by gastric tube, and different swabs (especially nasopharyngeal, ear and umbilical cord), as well as blood cultures were immediately performed for bacteriological investigations and for SARS-CoV-2 RT-PCR test. The newborn was breathing autonomously, had a good control of body temperature and blood sugar. She received a 10% glucose infusion for 48 h, and on the second day an enteral feeding by nasogastric tube was progressively introduced, using artificial milk formulas adapted to preterm babies. Prophylactic antibiotherapy (penicillin G and amikacin) was initiated, considering the risk of neonatal infections in prematurity. On postnatal day 3, the newborn baby presented jaundice, respiratory distress and a clinical picture of ulcerative enterocolitis. Hemocultures were found negative, but SARS-CoV-2 RT-PCR was positive in oropharyngeal swab and cultures of gastric liquid isolated multiresistant Citrobacter sp. and Enterobacter cloacae. A phototherapy was prescribed for 3 days and previous antibiotics were replaced by meropenem and vancomycin based on the antibiogram. Despite this treatment, the newborn died on Day 5 in a picture of severe neonatal sepsis. The postoperative follow-up of the mother was marked by a persistence of fever for 3 days, varying between 39 and 40 °C. Although haemocultures and urine cultures were sterile, antibiotic therapy was readjusted on postoperative Day 3 as for the newborn, with ceftriaxone replaced by meropenem. C-reactive protein (CRP) varied from 106.53 mg/l on admission to 186 mg/l on postoperative Day 1, falling to 21.93 mg/l on Day 5 and below 3 mg/l on Day 7. After 7 days of hospitalization, the patient’s condition was stable, with no fever nor respiratory symptoms. She was discharged from the hospital and sent back to the COVID-19 isolation Center. A control of the SARS-CoV-2 RT-PCR was negative on Day 13, she returned back home. The late postpartum up to 6 weeks was unremarkable, with no complication. No medical staff involved in this case was subsequently found to be infected with SARS-CoV-2. Discussion One year after the first cases of COVID-19, the disease continues spreading at striking speed in many countries worldwide. People get contaminated mainly through direct means (including respiratory droplets and physical contacts with carriers) or by indirect contacts with contaminated objects [16]. Vertical transmission of SARS-CoV-2 during pregnancy is another possible route of transmission [9], although further investigations are still needed to confirm this eventuality. In previously published case reports of neonatal SARS-CoV-2 infections, it was not well established if the contamination occurred during pregnancy or after birth, especially during the delivery process [17]. Peritoneal lesions associated with SARS CoV-2 were previously suspected in patients [18–20]. Recently, the transplacental transmission of SARS-CoV-2 was reported [21, 22] and a classification of the COVID-19 infection in pregnant women, foetuses and newborns was suggested [23]. This case-report highlights a number of facts which suggest a possible intrauterine transmission of SARS-CoV-2 infection. We recognize, however, the limitations of our means of exploration which would confirm the case. Furthermore, none of the medical staff involved was subsequently found to be infected by SARS-CoV-2. The intense inflammatory reaction of the uterus and foetal appendages suggest a direct effect of SARS-CoV-2 on placenta. Conclusion This case is one of the first documented cases of COVID-19 in pregnancy in sub-Saharan Africa. The intense inflammatory reaction of the uterus and foetal appendages suggest a direct effect of SARS-CoV-2 on placenta. We strongly suggest obstetricians to carefully examine the aspect of the peritoneum, viscera and foetal appendages in affected pregnant women. Abbreviations COVID-19Coronavirus disease 2019 DRCDemocratic Republic of the Congo HPGRBHôpital Provincial Général de Référence de Bukavu, Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements We are very grateful to all those persons who contributed to this work, especially Dr. BULAMBO ASOKA Blandine. Authors sincerely thank all the staff of Provincial referral Hospital of Bukavu for their collaboration for this case study. Authors’ contributions EKB: drafted the manuscript, reviewed the literature, followed up the patient and edited the final manuscript. GMM, SZM, PMK, EBM, DGM, MB and GBB edited and revised the manuscript. All the authors approved the final version of the manuscript. Funding This case report received no funding. Availability of data and materials Materials and data provided in this case study are available from the corresponding author on reasonable request. Ethics approval and consent to participate The publication of this case was approved by the Ethics committee of the Catholic University of Bukavu, DRC. Consent for publication Consent for publication of the clinical details and/or laboratory results was obtained from the patient. Competing interests The authors declare that they have no competing interests.	1,3,5-TRIMETHOXYBENZENE, ACETAMINOPHEN, AMIKACIN, AZITHROMYCIN ANHYDROUS, CEFTRIAXONE, MEROPENEM, PENICILLIN G, VANCOMYCIN HYDROCHLORIDE	DrugsGivenReaction	CC BY	33472696	19,184,030	2021-01-20
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Exposure during pregnancy'.	A case study of the first pregnant woman with COVID-19 in Bukavu, eastern Democratic Republic of the Congo. BACKGROUND Vertical transmission of covid-19 is possible; its risk factors are worth researching. The placental changes found in pregnant women have a definite impact on the foetus. METHODS We report a case of a 25-year-old woman, gravida 3, para 2 (2 alive children), with a history of two caesarean deliveries, who was infected by the SARS-CoV-2 during the last term of her pregnancy. She gave birth by caesarean at 34 weeks of gestation to a newborn baby also infected with SARS-CoV-2. The peri-operative observations noted several eruptive lesions in the pelvis, bleeding on contact. Microscopic examination of the foetal appendages revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels. CONCLUSIONS This case is one of the first documented cases of COVID-19 in pregnancy in sub-Saharan Africa. We strongly suggest obstetricians to carefully examine the aspect of the peritoneum, viscera and foetal appendages in affected pregnant women. Background In December 2019, first cases of severe acute respiratory syndrome (SARS) due to a new coronavirus (SARS-CoV-2) were reported from Wuhan in China. Soon after, the disease, subsequently named “the 2019 novel coronavirus disease” (COVID-19) and declared a pandemic by the World Health Organisation (WHO) [1], has resulted in over 18.9 million confirmed cases and more than 709,000 deaths worldwide [2]. The pregnant woman can be considered to be more at risk of severe form than the non pregnant woman [3]. Their fragile immunity and frequent comorbidities such as obesity, diabetes mellitus, arterial hypertension, or cardiovascular diseases may expose them at higher risks of developing severe forms of the disease [4] and to adverse pregnancy outcomes, especially during the third trimester [5]. COVID-19 causes pneumonia with acute respiratory distress syndrome (ARDS), which can compromise natural delivery, increase maternal morbidity, or even lead to maternal death [6]. Knowledge about coronavirus disease during pregnancy is still limited [4], and vertical transmission in utero is not yet well established [4, 5]. The risk of mother-to-child transmission of COVID-19 seems to be low [7–9]. Cases of perinatal transmission of COVID-19 have been described, but it is still unclear if this occurred via the transplacental or other routes during delivery [10]. Furthermore, COVID-19 may constitute a threat of premature delivery, intrauterine growth retardation, premature rupture of the membranes, in-utero foetal death or even a premature neonatal death during delivery or soon after [8]. Among pregnant women with SARS-CoV-2, preterm birth is reported 12.9% [11]. There are no established guidelines about the best timing nor the best mode of delivery in COVID-19 infected pregnant women to optimize foetal and maternal well-being [12]. A study on the association between mode of delivery and maternal and neonatal outcomes in COVID-19 patients in Spain has shown that caesarean section was associated with an increased risk for maternal clinical deterioration which remained significant after adjustment for confounders [13]. However, a few case-reports have shown a benefit of a caesarean section on the improvement of respiratory distress in severely affected patients [5, 12]. There have been only a few studies that have investigated the intraoperative findings in women undergoing caesarean delivery [14], or changes in the foetal appendages that may explain the risk of maternal-foetal transmission [15]. We report the first documented case of COVID-19 in a pregnant woman recorded in the province of South Kivu, in eastern Democratic Republic of the Congo (DRC) who gave birth by cesarean section to a premature newborn also infected by SARS-CoV-2. Her pelvic organs exhibited a particular inflammatory appearance, and fetal appendages revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels. Case presentation A 25-year-old woman, gravida 3 para 2, at 34 weeks of gestation, with no medical history of cardiovascular nor other chronic diseases, was admitted to the labour and delivery unit of the “Hôpital Provincial Général de Référence de Bukavu” (HPGRB), in South-Kivu, for preterm labour contractions in a context of COVID-19. She had an history of 2 previous caesarean sections (the first one due to a cervical dystocia and the second indicated because of the prior caesarean), her last born wasaged 16 months. Her husband, tested negative for SARS-CoV-2, was a contact person of a COVID-19 confirmed case. Three weeks before admission, she complained of fever, not responding to acetaminophen. Her obstetrician prescribed her antibiotics, anti-malaria, and anti-spasmodic drugs. Two weeks later, as fever persisted despite all these medications, a reverse transcriptase-polymerase chain reaction (RT-PCR) nasopharyngeal swabs was performed and confirmed she was infected by SARS-CoV-2. She was then admitted to the provincial COVID-19 treatment center for isolation and health care. Upon arrival to the center, her body temperature was 38.7 °C. Gynecologic examination was unremarkable. All bacteriological tests, including hemocultures and cultures of urines were negative. She received antipyretics (acetaminophen), antispasmodics trimethylphloroglucinol and antibiotics (oral azithromycin for 5 days and intravenous ceftriaxone). Two days later, she complained of hypogastric pain, like uterine contractions of low intensity. Obstetricians of the HPGRB were contacted and recommended the administration of antispasmodics intravenously in perfusion. Despite this treatments, fever and uterine contractions persisted, so intravenous dexamethasone 12 mg daily was administered for fetal pulmonary maturation, associated with a tocolysis using nifedipine for 48 h. As the frequency, intensity and duration of contractions increased, accompanied by cervical changes (dilation, effacement, softening, and movement to a more anterior position), the patient was transferred to the labour and delivery unit of the HPGRB for an optimal care. A rapid SARS-Cov-2 antigen test was performed and found to be negative. On admission at the HPGRB, the patient had a good general condition. Her temperature (36.5 °C) and blood pressure (120/60 mmHg) were normal. The uterine height was 29 cm, the fœtus was in cephalic presentation. On vaginal examination, the uterine cervix was softened, median, 5 mm long and had a 5 cm dilatation. Membranes were intact and the fœtal head was mobile. An obstetrical ultrasound confirmed the cephalic presentation and estimated the foetal weight at 1600 g. Foetal monitoring confirmed a foetal well-being, with a stable foetal cardiac rhythm around 140 beats per minute. Tocography showed two to three contractions per minute and an intensity of 50 to 60 mmHg. A diagnosis of intractable preterm labor in a COVID-19 patient with a history of iterative caesarean deliveries was made. A classic Caesarean section with a Pfannestiel incision was performed. The peritoneal cavity and uterus were found to be very inflamed. Fetal appendages as well as the bladder were strewn with eruptive, vesicular lesions bleeding on contact (Figs. 1 and 2). The amniotic fluid was opalescent. The placenta weighed 500 g and had a clot on the maternal side on less than 20% of the surface. Anatomopathological examination subsequently revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels (Figs. 3 and 4), and a diffuse hyalinization with marked angiogenesis of the villous stroma. Fig. 1 Peri-operative appearance showing several bleeding eruptive lesions on contact ( ) with the posterior surface of the uterus, on the ovaries, the left fallopian tube up to Douglas’ pouch Fig. 2 Same lesions in the right appendages of the uterus ( ) Fig. 3 Section of the placenta; in the Hematoxylin-eosin (HE) staining we noted on sections of the placenta at 100X magnification, highly vascularized villi, with vascular lesions ( ) in the form of congestion and thrombosis visualized in certain fields and large areas of stromal hyalinization Fig. 4 Cross section of the umbilical cord (Magnification: 40X, Hematoxylline-eosin staining). There is the presence of thrombus ( ) in the lumen of the umbilical vessel, Wharton’s jelly is without distinction, no inflammatory reaction About five minutes after skin incision, a female newborn weighing 1760 g was delivered with 1 and 5 min APGAR scores of 9–10. The newborn was immediately transferred to the neonatal ward for specialized neonatal treatment for an optimal care and to minimise the potential risk of infection. Gestational age was estimated at 33 weeks according to the Finnstrom score. The newborn received the usual care (drying, stimulation, vitamin K1, Fluorescein and care of the umbilical cord). A gastric liquid was collected by gastric tube, and different swabs (especially nasopharyngeal, ear and umbilical cord), as well as blood cultures were immediately performed for bacteriological investigations and for SARS-CoV-2 RT-PCR test. The newborn was breathing autonomously, had a good control of body temperature and blood sugar. She received a 10% glucose infusion for 48 h, and on the second day an enteral feeding by nasogastric tube was progressively introduced, using artificial milk formulas adapted to preterm babies. Prophylactic antibiotherapy (penicillin G and amikacin) was initiated, considering the risk of neonatal infections in prematurity. On postnatal day 3, the newborn baby presented jaundice, respiratory distress and a clinical picture of ulcerative enterocolitis. Hemocultures were found negative, but SARS-CoV-2 RT-PCR was positive in oropharyngeal swab and cultures of gastric liquid isolated multiresistant Citrobacter sp. and Enterobacter cloacae. A phototherapy was prescribed for 3 days and previous antibiotics were replaced by meropenem and vancomycin based on the antibiogram. Despite this treatment, the newborn died on Day 5 in a picture of severe neonatal sepsis. The postoperative follow-up of the mother was marked by a persistence of fever for 3 days, varying between 39 and 40 °C. Although haemocultures and urine cultures were sterile, antibiotic therapy was readjusted on postoperative Day 3 as for the newborn, with ceftriaxone replaced by meropenem. C-reactive protein (CRP) varied from 106.53 mg/l on admission to 186 mg/l on postoperative Day 1, falling to 21.93 mg/l on Day 5 and below 3 mg/l on Day 7. After 7 days of hospitalization, the patient’s condition was stable, with no fever nor respiratory symptoms. She was discharged from the hospital and sent back to the COVID-19 isolation Center. A control of the SARS-CoV-2 RT-PCR was negative on Day 13, she returned back home. The late postpartum up to 6 weeks was unremarkable, with no complication. No medical staff involved in this case was subsequently found to be infected with SARS-CoV-2. Discussion One year after the first cases of COVID-19, the disease continues spreading at striking speed in many countries worldwide. People get contaminated mainly through direct means (including respiratory droplets and physical contacts with carriers) or by indirect contacts with contaminated objects [16]. Vertical transmission of SARS-CoV-2 during pregnancy is another possible route of transmission [9], although further investigations are still needed to confirm this eventuality. In previously published case reports of neonatal SARS-CoV-2 infections, it was not well established if the contamination occurred during pregnancy or after birth, especially during the delivery process [17]. Peritoneal lesions associated with SARS CoV-2 were previously suspected in patients [18–20]. Recently, the transplacental transmission of SARS-CoV-2 was reported [21, 22] and a classification of the COVID-19 infection in pregnant women, foetuses and newborns was suggested [23]. This case-report highlights a number of facts which suggest a possible intrauterine transmission of SARS-CoV-2 infection. We recognize, however, the limitations of our means of exploration which would confirm the case. Furthermore, none of the medical staff involved was subsequently found to be infected by SARS-CoV-2. The intense inflammatory reaction of the uterus and foetal appendages suggest a direct effect of SARS-CoV-2 on placenta. Conclusion This case is one of the first documented cases of COVID-19 in pregnancy in sub-Saharan Africa. The intense inflammatory reaction of the uterus and foetal appendages suggest a direct effect of SARS-CoV-2 on placenta. We strongly suggest obstetricians to carefully examine the aspect of the peritoneum, viscera and foetal appendages in affected pregnant women. Abbreviations COVID-19Coronavirus disease 2019 DRCDemocratic Republic of the Congo HPGRBHôpital Provincial Général de Référence de Bukavu, Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements We are very grateful to all those persons who contributed to this work, especially Dr. BULAMBO ASOKA Blandine. Authors sincerely thank all the staff of Provincial referral Hospital of Bukavu for their collaboration for this case study. Authors’ contributions EKB: drafted the manuscript, reviewed the literature, followed up the patient and edited the final manuscript. GMM, SZM, PMK, EBM, DGM, MB and GBB edited and revised the manuscript. All the authors approved the final version of the manuscript. Funding This case report received no funding. Availability of data and materials Materials and data provided in this case study are available from the corresponding author on reasonable request. Ethics approval and consent to participate The publication of this case was approved by the Ethics committee of the Catholic University of Bukavu, DRC. Consent for publication Consent for publication of the clinical details and/or laboratory results was obtained from the patient. Competing interests The authors declare that they have no competing interests.	1,3,5-TRIMETHOXYBENZENE, ACETAMINOPHEN, AZITHROMYCIN ANHYDROUS, CEFTRIAXONE	DrugsGivenReaction	CC BY	33472696	19,183,964	2021-01-20
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Foetal exposure during pregnancy'.	A case study of the first pregnant woman with COVID-19 in Bukavu, eastern Democratic Republic of the Congo. BACKGROUND Vertical transmission of covid-19 is possible; its risk factors are worth researching. The placental changes found in pregnant women have a definite impact on the foetus. METHODS We report a case of a 25-year-old woman, gravida 3, para 2 (2 alive children), with a history of two caesarean deliveries, who was infected by the SARS-CoV-2 during the last term of her pregnancy. She gave birth by caesarean at 34 weeks of gestation to a newborn baby also infected with SARS-CoV-2. The peri-operative observations noted several eruptive lesions in the pelvis, bleeding on contact. Microscopic examination of the foetal appendages revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels. CONCLUSIONS This case is one of the first documented cases of COVID-19 in pregnancy in sub-Saharan Africa. We strongly suggest obstetricians to carefully examine the aspect of the peritoneum, viscera and foetal appendages in affected pregnant women. Background In December 2019, first cases of severe acute respiratory syndrome (SARS) due to a new coronavirus (SARS-CoV-2) were reported from Wuhan in China. Soon after, the disease, subsequently named “the 2019 novel coronavirus disease” (COVID-19) and declared a pandemic by the World Health Organisation (WHO) [1], has resulted in over 18.9 million confirmed cases and more than 709,000 deaths worldwide [2]. The pregnant woman can be considered to be more at risk of severe form than the non pregnant woman [3]. Their fragile immunity and frequent comorbidities such as obesity, diabetes mellitus, arterial hypertension, or cardiovascular diseases may expose them at higher risks of developing severe forms of the disease [4] and to adverse pregnancy outcomes, especially during the third trimester [5]. COVID-19 causes pneumonia with acute respiratory distress syndrome (ARDS), which can compromise natural delivery, increase maternal morbidity, or even lead to maternal death [6]. Knowledge about coronavirus disease during pregnancy is still limited [4], and vertical transmission in utero is not yet well established [4, 5]. The risk of mother-to-child transmission of COVID-19 seems to be low [7–9]. Cases of perinatal transmission of COVID-19 have been described, but it is still unclear if this occurred via the transplacental or other routes during delivery [10]. Furthermore, COVID-19 may constitute a threat of premature delivery, intrauterine growth retardation, premature rupture of the membranes, in-utero foetal death or even a premature neonatal death during delivery or soon after [8]. Among pregnant women with SARS-CoV-2, preterm birth is reported 12.9% [11]. There are no established guidelines about the best timing nor the best mode of delivery in COVID-19 infected pregnant women to optimize foetal and maternal well-being [12]. A study on the association between mode of delivery and maternal and neonatal outcomes in COVID-19 patients in Spain has shown that caesarean section was associated with an increased risk for maternal clinical deterioration which remained significant after adjustment for confounders [13]. However, a few case-reports have shown a benefit of a caesarean section on the improvement of respiratory distress in severely affected patients [5, 12]. There have been only a few studies that have investigated the intraoperative findings in women undergoing caesarean delivery [14], or changes in the foetal appendages that may explain the risk of maternal-foetal transmission [15]. We report the first documented case of COVID-19 in a pregnant woman recorded in the province of South Kivu, in eastern Democratic Republic of the Congo (DRC) who gave birth by cesarean section to a premature newborn also infected by SARS-CoV-2. Her pelvic organs exhibited a particular inflammatory appearance, and fetal appendages revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels. Case presentation A 25-year-old woman, gravida 3 para 2, at 34 weeks of gestation, with no medical history of cardiovascular nor other chronic diseases, was admitted to the labour and delivery unit of the “Hôpital Provincial Général de Référence de Bukavu” (HPGRB), in South-Kivu, for preterm labour contractions in a context of COVID-19. She had an history of 2 previous caesarean sections (the first one due to a cervical dystocia and the second indicated because of the prior caesarean), her last born wasaged 16 months. Her husband, tested negative for SARS-CoV-2, was a contact person of a COVID-19 confirmed case. Three weeks before admission, she complained of fever, not responding to acetaminophen. Her obstetrician prescribed her antibiotics, anti-malaria, and anti-spasmodic drugs. Two weeks later, as fever persisted despite all these medications, a reverse transcriptase-polymerase chain reaction (RT-PCR) nasopharyngeal swabs was performed and confirmed she was infected by SARS-CoV-2. She was then admitted to the provincial COVID-19 treatment center for isolation and health care. Upon arrival to the center, her body temperature was 38.7 °C. Gynecologic examination was unremarkable. All bacteriological tests, including hemocultures and cultures of urines were negative. She received antipyretics (acetaminophen), antispasmodics trimethylphloroglucinol and antibiotics (oral azithromycin for 5 days and intravenous ceftriaxone). Two days later, she complained of hypogastric pain, like uterine contractions of low intensity. Obstetricians of the HPGRB were contacted and recommended the administration of antispasmodics intravenously in perfusion. Despite this treatments, fever and uterine contractions persisted, so intravenous dexamethasone 12 mg daily was administered for fetal pulmonary maturation, associated with a tocolysis using nifedipine for 48 h. As the frequency, intensity and duration of contractions increased, accompanied by cervical changes (dilation, effacement, softening, and movement to a more anterior position), the patient was transferred to the labour and delivery unit of the HPGRB for an optimal care. A rapid SARS-Cov-2 antigen test was performed and found to be negative. On admission at the HPGRB, the patient had a good general condition. Her temperature (36.5 °C) and blood pressure (120/60 mmHg) were normal. The uterine height was 29 cm, the fœtus was in cephalic presentation. On vaginal examination, the uterine cervix was softened, median, 5 mm long and had a 5 cm dilatation. Membranes were intact and the fœtal head was mobile. An obstetrical ultrasound confirmed the cephalic presentation and estimated the foetal weight at 1600 g. Foetal monitoring confirmed a foetal well-being, with a stable foetal cardiac rhythm around 140 beats per minute. Tocography showed two to three contractions per minute and an intensity of 50 to 60 mmHg. A diagnosis of intractable preterm labor in a COVID-19 patient with a history of iterative caesarean deliveries was made. A classic Caesarean section with a Pfannestiel incision was performed. The peritoneal cavity and uterus were found to be very inflamed. Fetal appendages as well as the bladder were strewn with eruptive, vesicular lesions bleeding on contact (Figs. 1 and 2). The amniotic fluid was opalescent. The placenta weighed 500 g and had a clot on the maternal side on less than 20% of the surface. Anatomopathological examination subsequently revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels (Figs. 3 and 4), and a diffuse hyalinization with marked angiogenesis of the villous stroma. Fig. 1 Peri-operative appearance showing several bleeding eruptive lesions on contact ( ) with the posterior surface of the uterus, on the ovaries, the left fallopian tube up to Douglas’ pouch Fig. 2 Same lesions in the right appendages of the uterus ( ) Fig. 3 Section of the placenta; in the Hematoxylin-eosin (HE) staining we noted on sections of the placenta at 100X magnification, highly vascularized villi, with vascular lesions ( ) in the form of congestion and thrombosis visualized in certain fields and large areas of stromal hyalinization Fig. 4 Cross section of the umbilical cord (Magnification: 40X, Hematoxylline-eosin staining). There is the presence of thrombus ( ) in the lumen of the umbilical vessel, Wharton’s jelly is without distinction, no inflammatory reaction About five minutes after skin incision, a female newborn weighing 1760 g was delivered with 1 and 5 min APGAR scores of 9–10. The newborn was immediately transferred to the neonatal ward for specialized neonatal treatment for an optimal care and to minimise the potential risk of infection. Gestational age was estimated at 33 weeks according to the Finnstrom score. The newborn received the usual care (drying, stimulation, vitamin K1, Fluorescein and care of the umbilical cord). A gastric liquid was collected by gastric tube, and different swabs (especially nasopharyngeal, ear and umbilical cord), as well as blood cultures were immediately performed for bacteriological investigations and for SARS-CoV-2 RT-PCR test. The newborn was breathing autonomously, had a good control of body temperature and blood sugar. She received a 10% glucose infusion for 48 h, and on the second day an enteral feeding by nasogastric tube was progressively introduced, using artificial milk formulas adapted to preterm babies. Prophylactic antibiotherapy (penicillin G and amikacin) was initiated, considering the risk of neonatal infections in prematurity. On postnatal day 3, the newborn baby presented jaundice, respiratory distress and a clinical picture of ulcerative enterocolitis. Hemocultures were found negative, but SARS-CoV-2 RT-PCR was positive in oropharyngeal swab and cultures of gastric liquid isolated multiresistant Citrobacter sp. and Enterobacter cloacae. A phototherapy was prescribed for 3 days and previous antibiotics were replaced by meropenem and vancomycin based on the antibiogram. Despite this treatment, the newborn died on Day 5 in a picture of severe neonatal sepsis. The postoperative follow-up of the mother was marked by a persistence of fever for 3 days, varying between 39 and 40 °C. Although haemocultures and urine cultures were sterile, antibiotic therapy was readjusted on postoperative Day 3 as for the newborn, with ceftriaxone replaced by meropenem. C-reactive protein (CRP) varied from 106.53 mg/l on admission to 186 mg/l on postoperative Day 1, falling to 21.93 mg/l on Day 5 and below 3 mg/l on Day 7. After 7 days of hospitalization, the patient’s condition was stable, with no fever nor respiratory symptoms. She was discharged from the hospital and sent back to the COVID-19 isolation Center. A control of the SARS-CoV-2 RT-PCR was negative on Day 13, she returned back home. The late postpartum up to 6 weeks was unremarkable, with no complication. No medical staff involved in this case was subsequently found to be infected with SARS-CoV-2. Discussion One year after the first cases of COVID-19, the disease continues spreading at striking speed in many countries worldwide. People get contaminated mainly through direct means (including respiratory droplets and physical contacts with carriers) or by indirect contacts with contaminated objects [16]. Vertical transmission of SARS-CoV-2 during pregnancy is another possible route of transmission [9], although further investigations are still needed to confirm this eventuality. In previously published case reports of neonatal SARS-CoV-2 infections, it was not well established if the contamination occurred during pregnancy or after birth, especially during the delivery process [17]. Peritoneal lesions associated with SARS CoV-2 were previously suspected in patients [18–20]. Recently, the transplacental transmission of SARS-CoV-2 was reported [21, 22] and a classification of the COVID-19 infection in pregnant women, foetuses and newborns was suggested [23]. This case-report highlights a number of facts which suggest a possible intrauterine transmission of SARS-CoV-2 infection. We recognize, however, the limitations of our means of exploration which would confirm the case. Furthermore, none of the medical staff involved was subsequently found to be infected by SARS-CoV-2. The intense inflammatory reaction of the uterus and foetal appendages suggest a direct effect of SARS-CoV-2 on placenta. Conclusion This case is one of the first documented cases of COVID-19 in pregnancy in sub-Saharan Africa. The intense inflammatory reaction of the uterus and foetal appendages suggest a direct effect of SARS-CoV-2 on placenta. We strongly suggest obstetricians to carefully examine the aspect of the peritoneum, viscera and foetal appendages in affected pregnant women. Abbreviations COVID-19Coronavirus disease 2019 DRCDemocratic Republic of the Congo HPGRBHôpital Provincial Général de Référence de Bukavu, Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements We are very grateful to all those persons who contributed to this work, especially Dr. BULAMBO ASOKA Blandine. Authors sincerely thank all the staff of Provincial referral Hospital of Bukavu for their collaboration for this case study. Authors’ contributions EKB: drafted the manuscript, reviewed the literature, followed up the patient and edited the final manuscript. GMM, SZM, PMK, EBM, DGM, MB and GBB edited and revised the manuscript. All the authors approved the final version of the manuscript. Funding This case report received no funding. Availability of data and materials Materials and data provided in this case study are available from the corresponding author on reasonable request. Ethics approval and consent to participate The publication of this case was approved by the Ethics committee of the Catholic University of Bukavu, DRC. Consent for publication Consent for publication of the clinical details and/or laboratory results was obtained from the patient. Competing interests The authors declare that they have no competing interests.	1,3,5-TRIMETHOXYBENZENE, ACETAMINOPHEN, AMIKACIN, AZITHROMYCIN ANHYDROUS, CEFTRIAXONE, MEROPENEM, PENICILLIN G, VANCOMYCIN HYDROCHLORIDE	DrugsGivenReaction	CC BY	33472696	19,184,030	2021-01-20
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Off label use'.	A case study of the first pregnant woman with COVID-19 in Bukavu, eastern Democratic Republic of the Congo. BACKGROUND Vertical transmission of covid-19 is possible; its risk factors are worth researching. The placental changes found in pregnant women have a definite impact on the foetus. METHODS We report a case of a 25-year-old woman, gravida 3, para 2 (2 alive children), with a history of two caesarean deliveries, who was infected by the SARS-CoV-2 during the last term of her pregnancy. She gave birth by caesarean at 34 weeks of gestation to a newborn baby also infected with SARS-CoV-2. The peri-operative observations noted several eruptive lesions in the pelvis, bleeding on contact. Microscopic examination of the foetal appendages revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels. CONCLUSIONS This case is one of the first documented cases of COVID-19 in pregnancy in sub-Saharan Africa. We strongly suggest obstetricians to carefully examine the aspect of the peritoneum, viscera and foetal appendages in affected pregnant women. Background In December 2019, first cases of severe acute respiratory syndrome (SARS) due to a new coronavirus (SARS-CoV-2) were reported from Wuhan in China. Soon after, the disease, subsequently named “the 2019 novel coronavirus disease” (COVID-19) and declared a pandemic by the World Health Organisation (WHO) [1], has resulted in over 18.9 million confirmed cases and more than 709,000 deaths worldwide [2]. The pregnant woman can be considered to be more at risk of severe form than the non pregnant woman [3]. Their fragile immunity and frequent comorbidities such as obesity, diabetes mellitus, arterial hypertension, or cardiovascular diseases may expose them at higher risks of developing severe forms of the disease [4] and to adverse pregnancy outcomes, especially during the third trimester [5]. COVID-19 causes pneumonia with acute respiratory distress syndrome (ARDS), which can compromise natural delivery, increase maternal morbidity, or even lead to maternal death [6]. Knowledge about coronavirus disease during pregnancy is still limited [4], and vertical transmission in utero is not yet well established [4, 5]. The risk of mother-to-child transmission of COVID-19 seems to be low [7–9]. Cases of perinatal transmission of COVID-19 have been described, but it is still unclear if this occurred via the transplacental or other routes during delivery [10]. Furthermore, COVID-19 may constitute a threat of premature delivery, intrauterine growth retardation, premature rupture of the membranes, in-utero foetal death or even a premature neonatal death during delivery or soon after [8]. Among pregnant women with SARS-CoV-2, preterm birth is reported 12.9% [11]. There are no established guidelines about the best timing nor the best mode of delivery in COVID-19 infected pregnant women to optimize foetal and maternal well-being [12]. A study on the association between mode of delivery and maternal and neonatal outcomes in COVID-19 patients in Spain has shown that caesarean section was associated with an increased risk for maternal clinical deterioration which remained significant after adjustment for confounders [13]. However, a few case-reports have shown a benefit of a caesarean section on the improvement of respiratory distress in severely affected patients [5, 12]. There have been only a few studies that have investigated the intraoperative findings in women undergoing caesarean delivery [14], or changes in the foetal appendages that may explain the risk of maternal-foetal transmission [15]. We report the first documented case of COVID-19 in a pregnant woman recorded in the province of South Kivu, in eastern Democratic Republic of the Congo (DRC) who gave birth by cesarean section to a premature newborn also infected by SARS-CoV-2. Her pelvic organs exhibited a particular inflammatory appearance, and fetal appendages revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels. Case presentation A 25-year-old woman, gravida 3 para 2, at 34 weeks of gestation, with no medical history of cardiovascular nor other chronic diseases, was admitted to the labour and delivery unit of the “Hôpital Provincial Général de Référence de Bukavu” (HPGRB), in South-Kivu, for preterm labour contractions in a context of COVID-19. She had an history of 2 previous caesarean sections (the first one due to a cervical dystocia and the second indicated because of the prior caesarean), her last born wasaged 16 months. Her husband, tested negative for SARS-CoV-2, was a contact person of a COVID-19 confirmed case. Three weeks before admission, she complained of fever, not responding to acetaminophen. Her obstetrician prescribed her antibiotics, anti-malaria, and anti-spasmodic drugs. Two weeks later, as fever persisted despite all these medications, a reverse transcriptase-polymerase chain reaction (RT-PCR) nasopharyngeal swabs was performed and confirmed she was infected by SARS-CoV-2. She was then admitted to the provincial COVID-19 treatment center for isolation and health care. Upon arrival to the center, her body temperature was 38.7 °C. Gynecologic examination was unremarkable. All bacteriological tests, including hemocultures and cultures of urines were negative. She received antipyretics (acetaminophen), antispasmodics trimethylphloroglucinol and antibiotics (oral azithromycin for 5 days and intravenous ceftriaxone). Two days later, she complained of hypogastric pain, like uterine contractions of low intensity. Obstetricians of the HPGRB were contacted and recommended the administration of antispasmodics intravenously in perfusion. Despite this treatments, fever and uterine contractions persisted, so intravenous dexamethasone 12 mg daily was administered for fetal pulmonary maturation, associated with a tocolysis using nifedipine for 48 h. As the frequency, intensity and duration of contractions increased, accompanied by cervical changes (dilation, effacement, softening, and movement to a more anterior position), the patient was transferred to the labour and delivery unit of the HPGRB for an optimal care. A rapid SARS-Cov-2 antigen test was performed and found to be negative. On admission at the HPGRB, the patient had a good general condition. Her temperature (36.5 °C) and blood pressure (120/60 mmHg) were normal. The uterine height was 29 cm, the fœtus was in cephalic presentation. On vaginal examination, the uterine cervix was softened, median, 5 mm long and had a 5 cm dilatation. Membranes were intact and the fœtal head was mobile. An obstetrical ultrasound confirmed the cephalic presentation and estimated the foetal weight at 1600 g. Foetal monitoring confirmed a foetal well-being, with a stable foetal cardiac rhythm around 140 beats per minute. Tocography showed two to three contractions per minute and an intensity of 50 to 60 mmHg. A diagnosis of intractable preterm labor in a COVID-19 patient with a history of iterative caesarean deliveries was made. A classic Caesarean section with a Pfannestiel incision was performed. The peritoneal cavity and uterus were found to be very inflamed. Fetal appendages as well as the bladder were strewn with eruptive, vesicular lesions bleeding on contact (Figs. 1 and 2). The amniotic fluid was opalescent. The placenta weighed 500 g and had a clot on the maternal side on less than 20% of the surface. Anatomopathological examination subsequently revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels (Figs. 3 and 4), and a diffuse hyalinization with marked angiogenesis of the villous stroma. Fig. 1 Peri-operative appearance showing several bleeding eruptive lesions on contact ( ) with the posterior surface of the uterus, on the ovaries, the left fallopian tube up to Douglas’ pouch Fig. 2 Same lesions in the right appendages of the uterus ( ) Fig. 3 Section of the placenta; in the Hematoxylin-eosin (HE) staining we noted on sections of the placenta at 100X magnification, highly vascularized villi, with vascular lesions ( ) in the form of congestion and thrombosis visualized in certain fields and large areas of stromal hyalinization Fig. 4 Cross section of the umbilical cord (Magnification: 40X, Hematoxylline-eosin staining). There is the presence of thrombus ( ) in the lumen of the umbilical vessel, Wharton’s jelly is without distinction, no inflammatory reaction About five minutes after skin incision, a female newborn weighing 1760 g was delivered with 1 and 5 min APGAR scores of 9–10. The newborn was immediately transferred to the neonatal ward for specialized neonatal treatment for an optimal care and to minimise the potential risk of infection. Gestational age was estimated at 33 weeks according to the Finnstrom score. The newborn received the usual care (drying, stimulation, vitamin K1, Fluorescein and care of the umbilical cord). A gastric liquid was collected by gastric tube, and different swabs (especially nasopharyngeal, ear and umbilical cord), as well as blood cultures were immediately performed for bacteriological investigations and for SARS-CoV-2 RT-PCR test. The newborn was breathing autonomously, had a good control of body temperature and blood sugar. She received a 10% glucose infusion for 48 h, and on the second day an enteral feeding by nasogastric tube was progressively introduced, using artificial milk formulas adapted to preterm babies. Prophylactic antibiotherapy (penicillin G and amikacin) was initiated, considering the risk of neonatal infections in prematurity. On postnatal day 3, the newborn baby presented jaundice, respiratory distress and a clinical picture of ulcerative enterocolitis. Hemocultures were found negative, but SARS-CoV-2 RT-PCR was positive in oropharyngeal swab and cultures of gastric liquid isolated multiresistant Citrobacter sp. and Enterobacter cloacae. A phototherapy was prescribed for 3 days and previous antibiotics were replaced by meropenem and vancomycin based on the antibiogram. Despite this treatment, the newborn died on Day 5 in a picture of severe neonatal sepsis. The postoperative follow-up of the mother was marked by a persistence of fever for 3 days, varying between 39 and 40 °C. Although haemocultures and urine cultures were sterile, antibiotic therapy was readjusted on postoperative Day 3 as for the newborn, with ceftriaxone replaced by meropenem. C-reactive protein (CRP) varied from 106.53 mg/l on admission to 186 mg/l on postoperative Day 1, falling to 21.93 mg/l on Day 5 and below 3 mg/l on Day 7. After 7 days of hospitalization, the patient’s condition was stable, with no fever nor respiratory symptoms. She was discharged from the hospital and sent back to the COVID-19 isolation Center. A control of the SARS-CoV-2 RT-PCR was negative on Day 13, she returned back home. The late postpartum up to 6 weeks was unremarkable, with no complication. No medical staff involved in this case was subsequently found to be infected with SARS-CoV-2. Discussion One year after the first cases of COVID-19, the disease continues spreading at striking speed in many countries worldwide. People get contaminated mainly through direct means (including respiratory droplets and physical contacts with carriers) or by indirect contacts with contaminated objects [16]. Vertical transmission of SARS-CoV-2 during pregnancy is another possible route of transmission [9], although further investigations are still needed to confirm this eventuality. In previously published case reports of neonatal SARS-CoV-2 infections, it was not well established if the contamination occurred during pregnancy or after birth, especially during the delivery process [17]. Peritoneal lesions associated with SARS CoV-2 were previously suspected in patients [18–20]. Recently, the transplacental transmission of SARS-CoV-2 was reported [21, 22] and a classification of the COVID-19 infection in pregnant women, foetuses and newborns was suggested [23]. This case-report highlights a number of facts which suggest a possible intrauterine transmission of SARS-CoV-2 infection. We recognize, however, the limitations of our means of exploration which would confirm the case. Furthermore, none of the medical staff involved was subsequently found to be infected by SARS-CoV-2. The intense inflammatory reaction of the uterus and foetal appendages suggest a direct effect of SARS-CoV-2 on placenta. Conclusion This case is one of the first documented cases of COVID-19 in pregnancy in sub-Saharan Africa. The intense inflammatory reaction of the uterus and foetal appendages suggest a direct effect of SARS-CoV-2 on placenta. We strongly suggest obstetricians to carefully examine the aspect of the peritoneum, viscera and foetal appendages in affected pregnant women. Abbreviations COVID-19Coronavirus disease 2019 DRCDemocratic Republic of the Congo HPGRBHôpital Provincial Général de Référence de Bukavu, Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements We are very grateful to all those persons who contributed to this work, especially Dr. BULAMBO ASOKA Blandine. Authors sincerely thank all the staff of Provincial referral Hospital of Bukavu for their collaboration for this case study. Authors’ contributions EKB: drafted the manuscript, reviewed the literature, followed up the patient and edited the final manuscript. GMM, SZM, PMK, EBM, DGM, MB and GBB edited and revised the manuscript. All the authors approved the final version of the manuscript. Funding This case report received no funding. Availability of data and materials Materials and data provided in this case study are available from the corresponding author on reasonable request. Ethics approval and consent to participate The publication of this case was approved by the Ethics committee of the Catholic University of Bukavu, DRC. Consent for publication Consent for publication of the clinical details and/or laboratory results was obtained from the patient. Competing interests The authors declare that they have no competing interests.	1,3,5-TRIMETHOXYBENZENE, ACETAMINOPHEN, AZITHROMYCIN ANHYDROUS, CEFTRIAXONE	DrugsGivenReaction	CC BY	33472696	19,183,964	2021-01-20
What was the administration route of drug 'ACETAMINOPHEN'?	A case study of the first pregnant woman with COVID-19 in Bukavu, eastern Democratic Republic of the Congo. BACKGROUND Vertical transmission of covid-19 is possible; its risk factors are worth researching. The placental changes found in pregnant women have a definite impact on the foetus. METHODS We report a case of a 25-year-old woman, gravida 3, para 2 (2 alive children), with a history of two caesarean deliveries, who was infected by the SARS-CoV-2 during the last term of her pregnancy. She gave birth by caesarean at 34 weeks of gestation to a newborn baby also infected with SARS-CoV-2. The peri-operative observations noted several eruptive lesions in the pelvis, bleeding on contact. Microscopic examination of the foetal appendages revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels. CONCLUSIONS This case is one of the first documented cases of COVID-19 in pregnancy in sub-Saharan Africa. We strongly suggest obstetricians to carefully examine the aspect of the peritoneum, viscera and foetal appendages in affected pregnant women. Background In December 2019, first cases of severe acute respiratory syndrome (SARS) due to a new coronavirus (SARS-CoV-2) were reported from Wuhan in China. Soon after, the disease, subsequently named “the 2019 novel coronavirus disease” (COVID-19) and declared a pandemic by the World Health Organisation (WHO) [1], has resulted in over 18.9 million confirmed cases and more than 709,000 deaths worldwide [2]. The pregnant woman can be considered to be more at risk of severe form than the non pregnant woman [3]. Their fragile immunity and frequent comorbidities such as obesity, diabetes mellitus, arterial hypertension, or cardiovascular diseases may expose them at higher risks of developing severe forms of the disease [4] and to adverse pregnancy outcomes, especially during the third trimester [5]. COVID-19 causes pneumonia with acute respiratory distress syndrome (ARDS), which can compromise natural delivery, increase maternal morbidity, or even lead to maternal death [6]. Knowledge about coronavirus disease during pregnancy is still limited [4], and vertical transmission in utero is not yet well established [4, 5]. The risk of mother-to-child transmission of COVID-19 seems to be low [7–9]. Cases of perinatal transmission of COVID-19 have been described, but it is still unclear if this occurred via the transplacental or other routes during delivery [10]. Furthermore, COVID-19 may constitute a threat of premature delivery, intrauterine growth retardation, premature rupture of the membranes, in-utero foetal death or even a premature neonatal death during delivery or soon after [8]. Among pregnant women with SARS-CoV-2, preterm birth is reported 12.9% [11]. There are no established guidelines about the best timing nor the best mode of delivery in COVID-19 infected pregnant women to optimize foetal and maternal well-being [12]. A study on the association between mode of delivery and maternal and neonatal outcomes in COVID-19 patients in Spain has shown that caesarean section was associated with an increased risk for maternal clinical deterioration which remained significant after adjustment for confounders [13]. However, a few case-reports have shown a benefit of a caesarean section on the improvement of respiratory distress in severely affected patients [5, 12]. There have been only a few studies that have investigated the intraoperative findings in women undergoing caesarean delivery [14], or changes in the foetal appendages that may explain the risk of maternal-foetal transmission [15]. We report the first documented case of COVID-19 in a pregnant woman recorded in the province of South Kivu, in eastern Democratic Republic of the Congo (DRC) who gave birth by cesarean section to a premature newborn also infected by SARS-CoV-2. Her pelvic organs exhibited a particular inflammatory appearance, and fetal appendages revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels. Case presentation A 25-year-old woman, gravida 3 para 2, at 34 weeks of gestation, with no medical history of cardiovascular nor other chronic diseases, was admitted to the labour and delivery unit of the “Hôpital Provincial Général de Référence de Bukavu” (HPGRB), in South-Kivu, for preterm labour contractions in a context of COVID-19. She had an history of 2 previous caesarean sections (the first one due to a cervical dystocia and the second indicated because of the prior caesarean), her last born wasaged 16 months. Her husband, tested negative for SARS-CoV-2, was a contact person of a COVID-19 confirmed case. Three weeks before admission, she complained of fever, not responding to acetaminophen. Her obstetrician prescribed her antibiotics, anti-malaria, and anti-spasmodic drugs. Two weeks later, as fever persisted despite all these medications, a reverse transcriptase-polymerase chain reaction (RT-PCR) nasopharyngeal swabs was performed and confirmed she was infected by SARS-CoV-2. She was then admitted to the provincial COVID-19 treatment center for isolation and health care. Upon arrival to the center, her body temperature was 38.7 °C. Gynecologic examination was unremarkable. All bacteriological tests, including hemocultures and cultures of urines were negative. She received antipyretics (acetaminophen), antispasmodics trimethylphloroglucinol and antibiotics (oral azithromycin for 5 days and intravenous ceftriaxone). Two days later, she complained of hypogastric pain, like uterine contractions of low intensity. Obstetricians of the HPGRB were contacted and recommended the administration of antispasmodics intravenously in perfusion. Despite this treatments, fever and uterine contractions persisted, so intravenous dexamethasone 12 mg daily was administered for fetal pulmonary maturation, associated with a tocolysis using nifedipine for 48 h. As the frequency, intensity and duration of contractions increased, accompanied by cervical changes (dilation, effacement, softening, and movement to a more anterior position), the patient was transferred to the labour and delivery unit of the HPGRB for an optimal care. A rapid SARS-Cov-2 antigen test was performed and found to be negative. On admission at the HPGRB, the patient had a good general condition. Her temperature (36.5 °C) and blood pressure (120/60 mmHg) were normal. The uterine height was 29 cm, the fœtus was in cephalic presentation. On vaginal examination, the uterine cervix was softened, median, 5 mm long and had a 5 cm dilatation. Membranes were intact and the fœtal head was mobile. An obstetrical ultrasound confirmed the cephalic presentation and estimated the foetal weight at 1600 g. Foetal monitoring confirmed a foetal well-being, with a stable foetal cardiac rhythm around 140 beats per minute. Tocography showed two to three contractions per minute and an intensity of 50 to 60 mmHg. A diagnosis of intractable preterm labor in a COVID-19 patient with a history of iterative caesarean deliveries was made. A classic Caesarean section with a Pfannestiel incision was performed. The peritoneal cavity and uterus were found to be very inflamed. Fetal appendages as well as the bladder were strewn with eruptive, vesicular lesions bleeding on contact (Figs. 1 and 2). The amniotic fluid was opalescent. The placenta weighed 500 g and had a clot on the maternal side on less than 20% of the surface. Anatomopathological examination subsequently revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels (Figs. 3 and 4), and a diffuse hyalinization with marked angiogenesis of the villous stroma. Fig. 1 Peri-operative appearance showing several bleeding eruptive lesions on contact ( ) with the posterior surface of the uterus, on the ovaries, the left fallopian tube up to Douglas’ pouch Fig. 2 Same lesions in the right appendages of the uterus ( ) Fig. 3 Section of the placenta; in the Hematoxylin-eosin (HE) staining we noted on sections of the placenta at 100X magnification, highly vascularized villi, with vascular lesions ( ) in the form of congestion and thrombosis visualized in certain fields and large areas of stromal hyalinization Fig. 4 Cross section of the umbilical cord (Magnification: 40X, Hematoxylline-eosin staining). There is the presence of thrombus ( ) in the lumen of the umbilical vessel, Wharton’s jelly is without distinction, no inflammatory reaction About five minutes after skin incision, a female newborn weighing 1760 g was delivered with 1 and 5 min APGAR scores of 9–10. The newborn was immediately transferred to the neonatal ward for specialized neonatal treatment for an optimal care and to minimise the potential risk of infection. Gestational age was estimated at 33 weeks according to the Finnstrom score. The newborn received the usual care (drying, stimulation, vitamin K1, Fluorescein and care of the umbilical cord). A gastric liquid was collected by gastric tube, and different swabs (especially nasopharyngeal, ear and umbilical cord), as well as blood cultures were immediately performed for bacteriological investigations and for SARS-CoV-2 RT-PCR test. The newborn was breathing autonomously, had a good control of body temperature and blood sugar. She received a 10% glucose infusion for 48 h, and on the second day an enteral feeding by nasogastric tube was progressively introduced, using artificial milk formulas adapted to preterm babies. Prophylactic antibiotherapy (penicillin G and amikacin) was initiated, considering the risk of neonatal infections in prematurity. On postnatal day 3, the newborn baby presented jaundice, respiratory distress and a clinical picture of ulcerative enterocolitis. Hemocultures were found negative, but SARS-CoV-2 RT-PCR was positive in oropharyngeal swab and cultures of gastric liquid isolated multiresistant Citrobacter sp. and Enterobacter cloacae. A phototherapy was prescribed for 3 days and previous antibiotics were replaced by meropenem and vancomycin based on the antibiogram. Despite this treatment, the newborn died on Day 5 in a picture of severe neonatal sepsis. The postoperative follow-up of the mother was marked by a persistence of fever for 3 days, varying between 39 and 40 °C. Although haemocultures and urine cultures were sterile, antibiotic therapy was readjusted on postoperative Day 3 as for the newborn, with ceftriaxone replaced by meropenem. C-reactive protein (CRP) varied from 106.53 mg/l on admission to 186 mg/l on postoperative Day 1, falling to 21.93 mg/l on Day 5 and below 3 mg/l on Day 7. After 7 days of hospitalization, the patient’s condition was stable, with no fever nor respiratory symptoms. She was discharged from the hospital and sent back to the COVID-19 isolation Center. A control of the SARS-CoV-2 RT-PCR was negative on Day 13, she returned back home. The late postpartum up to 6 weeks was unremarkable, with no complication. No medical staff involved in this case was subsequently found to be infected with SARS-CoV-2. Discussion One year after the first cases of COVID-19, the disease continues spreading at striking speed in many countries worldwide. People get contaminated mainly through direct means (including respiratory droplets and physical contacts with carriers) or by indirect contacts with contaminated objects [16]. Vertical transmission of SARS-CoV-2 during pregnancy is another possible route of transmission [9], although further investigations are still needed to confirm this eventuality. In previously published case reports of neonatal SARS-CoV-2 infections, it was not well established if the contamination occurred during pregnancy or after birth, especially during the delivery process [17]. Peritoneal lesions associated with SARS CoV-2 were previously suspected in patients [18–20]. Recently, the transplacental transmission of SARS-CoV-2 was reported [21, 22] and a classification of the COVID-19 infection in pregnant women, foetuses and newborns was suggested [23]. This case-report highlights a number of facts which suggest a possible intrauterine transmission of SARS-CoV-2 infection. We recognize, however, the limitations of our means of exploration which would confirm the case. Furthermore, none of the medical staff involved was subsequently found to be infected by SARS-CoV-2. The intense inflammatory reaction of the uterus and foetal appendages suggest a direct effect of SARS-CoV-2 on placenta. Conclusion This case is one of the first documented cases of COVID-19 in pregnancy in sub-Saharan Africa. The intense inflammatory reaction of the uterus and foetal appendages suggest a direct effect of SARS-CoV-2 on placenta. We strongly suggest obstetricians to carefully examine the aspect of the peritoneum, viscera and foetal appendages in affected pregnant women. Abbreviations COVID-19Coronavirus disease 2019 DRCDemocratic Republic of the Congo HPGRBHôpital Provincial Général de Référence de Bukavu, Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements We are very grateful to all those persons who contributed to this work, especially Dr. BULAMBO ASOKA Blandine. Authors sincerely thank all the staff of Provincial referral Hospital of Bukavu for their collaboration for this case study. Authors’ contributions EKB: drafted the manuscript, reviewed the literature, followed up the patient and edited the final manuscript. GMM, SZM, PMK, EBM, DGM, MB and GBB edited and revised the manuscript. All the authors approved the final version of the manuscript. Funding This case report received no funding. Availability of data and materials Materials and data provided in this case study are available from the corresponding author on reasonable request. Ethics approval and consent to participate The publication of this case was approved by the Ethics committee of the Catholic University of Bukavu, DRC. Consent for publication Consent for publication of the clinical details and/or laboratory results was obtained from the patient. Competing interests The authors declare that they have no competing interests.	Transplacental	DrugAdministrationRoute	CC BY	33472696	19,184,030	2021-01-20
What was the administration route of drug 'AZITHROMYCIN ANHYDROUS'?	A case study of the first pregnant woman with COVID-19 in Bukavu, eastern Democratic Republic of the Congo. BACKGROUND Vertical transmission of covid-19 is possible; its risk factors are worth researching. The placental changes found in pregnant women have a definite impact on the foetus. METHODS We report a case of a 25-year-old woman, gravida 3, para 2 (2 alive children), with a history of two caesarean deliveries, who was infected by the SARS-CoV-2 during the last term of her pregnancy. She gave birth by caesarean at 34 weeks of gestation to a newborn baby also infected with SARS-CoV-2. The peri-operative observations noted several eruptive lesions in the pelvis, bleeding on contact. Microscopic examination of the foetal appendages revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels. CONCLUSIONS This case is one of the first documented cases of COVID-19 in pregnancy in sub-Saharan Africa. We strongly suggest obstetricians to carefully examine the aspect of the peritoneum, viscera and foetal appendages in affected pregnant women. Background In December 2019, first cases of severe acute respiratory syndrome (SARS) due to a new coronavirus (SARS-CoV-2) were reported from Wuhan in China. Soon after, the disease, subsequently named “the 2019 novel coronavirus disease” (COVID-19) and declared a pandemic by the World Health Organisation (WHO) [1], has resulted in over 18.9 million confirmed cases and more than 709,000 deaths worldwide [2]. The pregnant woman can be considered to be more at risk of severe form than the non pregnant woman [3]. Their fragile immunity and frequent comorbidities such as obesity, diabetes mellitus, arterial hypertension, or cardiovascular diseases may expose them at higher risks of developing severe forms of the disease [4] and to adverse pregnancy outcomes, especially during the third trimester [5]. COVID-19 causes pneumonia with acute respiratory distress syndrome (ARDS), which can compromise natural delivery, increase maternal morbidity, or even lead to maternal death [6]. Knowledge about coronavirus disease during pregnancy is still limited [4], and vertical transmission in utero is not yet well established [4, 5]. The risk of mother-to-child transmission of COVID-19 seems to be low [7–9]. Cases of perinatal transmission of COVID-19 have been described, but it is still unclear if this occurred via the transplacental or other routes during delivery [10]. Furthermore, COVID-19 may constitute a threat of premature delivery, intrauterine growth retardation, premature rupture of the membranes, in-utero foetal death or even a premature neonatal death during delivery or soon after [8]. Among pregnant women with SARS-CoV-2, preterm birth is reported 12.9% [11]. There are no established guidelines about the best timing nor the best mode of delivery in COVID-19 infected pregnant women to optimize foetal and maternal well-being [12]. A study on the association between mode of delivery and maternal and neonatal outcomes in COVID-19 patients in Spain has shown that caesarean section was associated with an increased risk for maternal clinical deterioration which remained significant after adjustment for confounders [13]. However, a few case-reports have shown a benefit of a caesarean section on the improvement of respiratory distress in severely affected patients [5, 12]. There have been only a few studies that have investigated the intraoperative findings in women undergoing caesarean delivery [14], or changes in the foetal appendages that may explain the risk of maternal-foetal transmission [15]. We report the first documented case of COVID-19 in a pregnant woman recorded in the province of South Kivu, in eastern Democratic Republic of the Congo (DRC) who gave birth by cesarean section to a premature newborn also infected by SARS-CoV-2. Her pelvic organs exhibited a particular inflammatory appearance, and fetal appendages revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels. Case presentation A 25-year-old woman, gravida 3 para 2, at 34 weeks of gestation, with no medical history of cardiovascular nor other chronic diseases, was admitted to the labour and delivery unit of the “Hôpital Provincial Général de Référence de Bukavu” (HPGRB), in South-Kivu, for preterm labour contractions in a context of COVID-19. She had an history of 2 previous caesarean sections (the first one due to a cervical dystocia and the second indicated because of the prior caesarean), her last born wasaged 16 months. Her husband, tested negative for SARS-CoV-2, was a contact person of a COVID-19 confirmed case. Three weeks before admission, she complained of fever, not responding to acetaminophen. Her obstetrician prescribed her antibiotics, anti-malaria, and anti-spasmodic drugs. Two weeks later, as fever persisted despite all these medications, a reverse transcriptase-polymerase chain reaction (RT-PCR) nasopharyngeal swabs was performed and confirmed she was infected by SARS-CoV-2. She was then admitted to the provincial COVID-19 treatment center for isolation and health care. Upon arrival to the center, her body temperature was 38.7 °C. Gynecologic examination was unremarkable. All bacteriological tests, including hemocultures and cultures of urines were negative. She received antipyretics (acetaminophen), antispasmodics trimethylphloroglucinol and antibiotics (oral azithromycin for 5 days and intravenous ceftriaxone). Two days later, she complained of hypogastric pain, like uterine contractions of low intensity. Obstetricians of the HPGRB were contacted and recommended the administration of antispasmodics intravenously in perfusion. Despite this treatments, fever and uterine contractions persisted, so intravenous dexamethasone 12 mg daily was administered for fetal pulmonary maturation, associated with a tocolysis using nifedipine for 48 h. As the frequency, intensity and duration of contractions increased, accompanied by cervical changes (dilation, effacement, softening, and movement to a more anterior position), the patient was transferred to the labour and delivery unit of the HPGRB for an optimal care. A rapid SARS-Cov-2 antigen test was performed and found to be negative. On admission at the HPGRB, the patient had a good general condition. Her temperature (36.5 °C) and blood pressure (120/60 mmHg) were normal. The uterine height was 29 cm, the fœtus was in cephalic presentation. On vaginal examination, the uterine cervix was softened, median, 5 mm long and had a 5 cm dilatation. Membranes were intact and the fœtal head was mobile. An obstetrical ultrasound confirmed the cephalic presentation and estimated the foetal weight at 1600 g. Foetal monitoring confirmed a foetal well-being, with a stable foetal cardiac rhythm around 140 beats per minute. Tocography showed two to three contractions per minute and an intensity of 50 to 60 mmHg. A diagnosis of intractable preterm labor in a COVID-19 patient with a history of iterative caesarean deliveries was made. A classic Caesarean section with a Pfannestiel incision was performed. The peritoneal cavity and uterus were found to be very inflamed. Fetal appendages as well as the bladder were strewn with eruptive, vesicular lesions bleeding on contact (Figs. 1 and 2). The amniotic fluid was opalescent. The placenta weighed 500 g and had a clot on the maternal side on less than 20% of the surface. Anatomopathological examination subsequently revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels (Figs. 3 and 4), and a diffuse hyalinization with marked angiogenesis of the villous stroma. Fig. 1 Peri-operative appearance showing several bleeding eruptive lesions on contact ( ) with the posterior surface of the uterus, on the ovaries, the left fallopian tube up to Douglas’ pouch Fig. 2 Same lesions in the right appendages of the uterus ( ) Fig. 3 Section of the placenta; in the Hematoxylin-eosin (HE) staining we noted on sections of the placenta at 100X magnification, highly vascularized villi, with vascular lesions ( ) in the form of congestion and thrombosis visualized in certain fields and large areas of stromal hyalinization Fig. 4 Cross section of the umbilical cord (Magnification: 40X, Hematoxylline-eosin staining). There is the presence of thrombus ( ) in the lumen of the umbilical vessel, Wharton’s jelly is without distinction, no inflammatory reaction About five minutes after skin incision, a female newborn weighing 1760 g was delivered with 1 and 5 min APGAR scores of 9–10. The newborn was immediately transferred to the neonatal ward for specialized neonatal treatment for an optimal care and to minimise the potential risk of infection. Gestational age was estimated at 33 weeks according to the Finnstrom score. The newborn received the usual care (drying, stimulation, vitamin K1, Fluorescein and care of the umbilical cord). A gastric liquid was collected by gastric tube, and different swabs (especially nasopharyngeal, ear and umbilical cord), as well as blood cultures were immediately performed for bacteriological investigations and for SARS-CoV-2 RT-PCR test. The newborn was breathing autonomously, had a good control of body temperature and blood sugar. She received a 10% glucose infusion for 48 h, and on the second day an enteral feeding by nasogastric tube was progressively introduced, using artificial milk formulas adapted to preterm babies. Prophylactic antibiotherapy (penicillin G and amikacin) was initiated, considering the risk of neonatal infections in prematurity. On postnatal day 3, the newborn baby presented jaundice, respiratory distress and a clinical picture of ulcerative enterocolitis. Hemocultures were found negative, but SARS-CoV-2 RT-PCR was positive in oropharyngeal swab and cultures of gastric liquid isolated multiresistant Citrobacter sp. and Enterobacter cloacae. A phototherapy was prescribed for 3 days and previous antibiotics were replaced by meropenem and vancomycin based on the antibiogram. Despite this treatment, the newborn died on Day 5 in a picture of severe neonatal sepsis. The postoperative follow-up of the mother was marked by a persistence of fever for 3 days, varying between 39 and 40 °C. Although haemocultures and urine cultures were sterile, antibiotic therapy was readjusted on postoperative Day 3 as for the newborn, with ceftriaxone replaced by meropenem. C-reactive protein (CRP) varied from 106.53 mg/l on admission to 186 mg/l on postoperative Day 1, falling to 21.93 mg/l on Day 5 and below 3 mg/l on Day 7. After 7 days of hospitalization, the patient’s condition was stable, with no fever nor respiratory symptoms. She was discharged from the hospital and sent back to the COVID-19 isolation Center. A control of the SARS-CoV-2 RT-PCR was negative on Day 13, she returned back home. The late postpartum up to 6 weeks was unremarkable, with no complication. No medical staff involved in this case was subsequently found to be infected with SARS-CoV-2. Discussion One year after the first cases of COVID-19, the disease continues spreading at striking speed in many countries worldwide. People get contaminated mainly through direct means (including respiratory droplets and physical contacts with carriers) or by indirect contacts with contaminated objects [16]. Vertical transmission of SARS-CoV-2 during pregnancy is another possible route of transmission [9], although further investigations are still needed to confirm this eventuality. In previously published case reports of neonatal SARS-CoV-2 infections, it was not well established if the contamination occurred during pregnancy or after birth, especially during the delivery process [17]. Peritoneal lesions associated with SARS CoV-2 were previously suspected in patients [18–20]. Recently, the transplacental transmission of SARS-CoV-2 was reported [21, 22] and a classification of the COVID-19 infection in pregnant women, foetuses and newborns was suggested [23]. This case-report highlights a number of facts which suggest a possible intrauterine transmission of SARS-CoV-2 infection. We recognize, however, the limitations of our means of exploration which would confirm the case. Furthermore, none of the medical staff involved was subsequently found to be infected by SARS-CoV-2. The intense inflammatory reaction of the uterus and foetal appendages suggest a direct effect of SARS-CoV-2 on placenta. Conclusion This case is one of the first documented cases of COVID-19 in pregnancy in sub-Saharan Africa. The intense inflammatory reaction of the uterus and foetal appendages suggest a direct effect of SARS-CoV-2 on placenta. We strongly suggest obstetricians to carefully examine the aspect of the peritoneum, viscera and foetal appendages in affected pregnant women. Abbreviations COVID-19Coronavirus disease 2019 DRCDemocratic Republic of the Congo HPGRBHôpital Provincial Général de Référence de Bukavu, Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements We are very grateful to all those persons who contributed to this work, especially Dr. BULAMBO ASOKA Blandine. Authors sincerely thank all the staff of Provincial referral Hospital of Bukavu for their collaboration for this case study. Authors’ contributions EKB: drafted the manuscript, reviewed the literature, followed up the patient and edited the final manuscript. GMM, SZM, PMK, EBM, DGM, MB and GBB edited and revised the manuscript. All the authors approved the final version of the manuscript. Funding This case report received no funding. Availability of data and materials Materials and data provided in this case study are available from the corresponding author on reasonable request. Ethics approval and consent to participate The publication of this case was approved by the Ethics committee of the Catholic University of Bukavu, DRC. Consent for publication Consent for publication of the clinical details and/or laboratory results was obtained from the patient. Competing interests The authors declare that they have no competing interests.	Transplacental	DrugAdministrationRoute	CC BY	33472696	19,184,030	2021-01-20
What was the administration route of drug 'MEROPENEM'?	A case study of the first pregnant woman with COVID-19 in Bukavu, eastern Democratic Republic of the Congo. BACKGROUND Vertical transmission of covid-19 is possible; its risk factors are worth researching. The placental changes found in pregnant women have a definite impact on the foetus. METHODS We report a case of a 25-year-old woman, gravida 3, para 2 (2 alive children), with a history of two caesarean deliveries, who was infected by the SARS-CoV-2 during the last term of her pregnancy. She gave birth by caesarean at 34 weeks of gestation to a newborn baby also infected with SARS-CoV-2. The peri-operative observations noted several eruptive lesions in the pelvis, bleeding on contact. Microscopic examination of the foetal appendages revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels. CONCLUSIONS This case is one of the first documented cases of COVID-19 in pregnancy in sub-Saharan Africa. We strongly suggest obstetricians to carefully examine the aspect of the peritoneum, viscera and foetal appendages in affected pregnant women. Background In December 2019, first cases of severe acute respiratory syndrome (SARS) due to a new coronavirus (SARS-CoV-2) were reported from Wuhan in China. Soon after, the disease, subsequently named “the 2019 novel coronavirus disease” (COVID-19) and declared a pandemic by the World Health Organisation (WHO) [1], has resulted in over 18.9 million confirmed cases and more than 709,000 deaths worldwide [2]. The pregnant woman can be considered to be more at risk of severe form than the non pregnant woman [3]. Their fragile immunity and frequent comorbidities such as obesity, diabetes mellitus, arterial hypertension, or cardiovascular diseases may expose them at higher risks of developing severe forms of the disease [4] and to adverse pregnancy outcomes, especially during the third trimester [5]. COVID-19 causes pneumonia with acute respiratory distress syndrome (ARDS), which can compromise natural delivery, increase maternal morbidity, or even lead to maternal death [6]. Knowledge about coronavirus disease during pregnancy is still limited [4], and vertical transmission in utero is not yet well established [4, 5]. The risk of mother-to-child transmission of COVID-19 seems to be low [7–9]. Cases of perinatal transmission of COVID-19 have been described, but it is still unclear if this occurred via the transplacental or other routes during delivery [10]. Furthermore, COVID-19 may constitute a threat of premature delivery, intrauterine growth retardation, premature rupture of the membranes, in-utero foetal death or even a premature neonatal death during delivery or soon after [8]. Among pregnant women with SARS-CoV-2, preterm birth is reported 12.9% [11]. There are no established guidelines about the best timing nor the best mode of delivery in COVID-19 infected pregnant women to optimize foetal and maternal well-being [12]. A study on the association between mode of delivery and maternal and neonatal outcomes in COVID-19 patients in Spain has shown that caesarean section was associated with an increased risk for maternal clinical deterioration which remained significant after adjustment for confounders [13]. However, a few case-reports have shown a benefit of a caesarean section on the improvement of respiratory distress in severely affected patients [5, 12]. There have been only a few studies that have investigated the intraoperative findings in women undergoing caesarean delivery [14], or changes in the foetal appendages that may explain the risk of maternal-foetal transmission [15]. We report the first documented case of COVID-19 in a pregnant woman recorded in the province of South Kivu, in eastern Democratic Republic of the Congo (DRC) who gave birth by cesarean section to a premature newborn also infected by SARS-CoV-2. Her pelvic organs exhibited a particular inflammatory appearance, and fetal appendages revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels. Case presentation A 25-year-old woman, gravida 3 para 2, at 34 weeks of gestation, with no medical history of cardiovascular nor other chronic diseases, was admitted to the labour and delivery unit of the “Hôpital Provincial Général de Référence de Bukavu” (HPGRB), in South-Kivu, for preterm labour contractions in a context of COVID-19. She had an history of 2 previous caesarean sections (the first one due to a cervical dystocia and the second indicated because of the prior caesarean), her last born wasaged 16 months. Her husband, tested negative for SARS-CoV-2, was a contact person of a COVID-19 confirmed case. Three weeks before admission, she complained of fever, not responding to acetaminophen. Her obstetrician prescribed her antibiotics, anti-malaria, and anti-spasmodic drugs. Two weeks later, as fever persisted despite all these medications, a reverse transcriptase-polymerase chain reaction (RT-PCR) nasopharyngeal swabs was performed and confirmed she was infected by SARS-CoV-2. She was then admitted to the provincial COVID-19 treatment center for isolation and health care. Upon arrival to the center, her body temperature was 38.7 °C. Gynecologic examination was unremarkable. All bacteriological tests, including hemocultures and cultures of urines were negative. She received antipyretics (acetaminophen), antispasmodics trimethylphloroglucinol and antibiotics (oral azithromycin for 5 days and intravenous ceftriaxone). Two days later, she complained of hypogastric pain, like uterine contractions of low intensity. Obstetricians of the HPGRB were contacted and recommended the administration of antispasmodics intravenously in perfusion. Despite this treatments, fever and uterine contractions persisted, so intravenous dexamethasone 12 mg daily was administered for fetal pulmonary maturation, associated with a tocolysis using nifedipine for 48 h. As the frequency, intensity and duration of contractions increased, accompanied by cervical changes (dilation, effacement, softening, and movement to a more anterior position), the patient was transferred to the labour and delivery unit of the HPGRB for an optimal care. A rapid SARS-Cov-2 antigen test was performed and found to be negative. On admission at the HPGRB, the patient had a good general condition. Her temperature (36.5 °C) and blood pressure (120/60 mmHg) were normal. The uterine height was 29 cm, the fœtus was in cephalic presentation. On vaginal examination, the uterine cervix was softened, median, 5 mm long and had a 5 cm dilatation. Membranes were intact and the fœtal head was mobile. An obstetrical ultrasound confirmed the cephalic presentation and estimated the foetal weight at 1600 g. Foetal monitoring confirmed a foetal well-being, with a stable foetal cardiac rhythm around 140 beats per minute. Tocography showed two to three contractions per minute and an intensity of 50 to 60 mmHg. A diagnosis of intractable preterm labor in a COVID-19 patient with a history of iterative caesarean deliveries was made. A classic Caesarean section with a Pfannestiel incision was performed. The peritoneal cavity and uterus were found to be very inflamed. Fetal appendages as well as the bladder were strewn with eruptive, vesicular lesions bleeding on contact (Figs. 1 and 2). The amniotic fluid was opalescent. The placenta weighed 500 g and had a clot on the maternal side on less than 20% of the surface. Anatomopathological examination subsequently revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels (Figs. 3 and 4), and a diffuse hyalinization with marked angiogenesis of the villous stroma. Fig. 1 Peri-operative appearance showing several bleeding eruptive lesions on contact ( ) with the posterior surface of the uterus, on the ovaries, the left fallopian tube up to Douglas’ pouch Fig. 2 Same lesions in the right appendages of the uterus ( ) Fig. 3 Section of the placenta; in the Hematoxylin-eosin (HE) staining we noted on sections of the placenta at 100X magnification, highly vascularized villi, with vascular lesions ( ) in the form of congestion and thrombosis visualized in certain fields and large areas of stromal hyalinization Fig. 4 Cross section of the umbilical cord (Magnification: 40X, Hematoxylline-eosin staining). There is the presence of thrombus ( ) in the lumen of the umbilical vessel, Wharton’s jelly is without distinction, no inflammatory reaction About five minutes after skin incision, a female newborn weighing 1760 g was delivered with 1 and 5 min APGAR scores of 9–10. The newborn was immediately transferred to the neonatal ward for specialized neonatal treatment for an optimal care and to minimise the potential risk of infection. Gestational age was estimated at 33 weeks according to the Finnstrom score. The newborn received the usual care (drying, stimulation, vitamin K1, Fluorescein and care of the umbilical cord). A gastric liquid was collected by gastric tube, and different swabs (especially nasopharyngeal, ear and umbilical cord), as well as blood cultures were immediately performed for bacteriological investigations and for SARS-CoV-2 RT-PCR test. The newborn was breathing autonomously, had a good control of body temperature and blood sugar. She received a 10% glucose infusion for 48 h, and on the second day an enteral feeding by nasogastric tube was progressively introduced, using artificial milk formulas adapted to preterm babies. Prophylactic antibiotherapy (penicillin G and amikacin) was initiated, considering the risk of neonatal infections in prematurity. On postnatal day 3, the newborn baby presented jaundice, respiratory distress and a clinical picture of ulcerative enterocolitis. Hemocultures were found negative, but SARS-CoV-2 RT-PCR was positive in oropharyngeal swab and cultures of gastric liquid isolated multiresistant Citrobacter sp. and Enterobacter cloacae. A phototherapy was prescribed for 3 days and previous antibiotics were replaced by meropenem and vancomycin based on the antibiogram. Despite this treatment, the newborn died on Day 5 in a picture of severe neonatal sepsis. The postoperative follow-up of the mother was marked by a persistence of fever for 3 days, varying between 39 and 40 °C. Although haemocultures and urine cultures were sterile, antibiotic therapy was readjusted on postoperative Day 3 as for the newborn, with ceftriaxone replaced by meropenem. C-reactive protein (CRP) varied from 106.53 mg/l on admission to 186 mg/l on postoperative Day 1, falling to 21.93 mg/l on Day 5 and below 3 mg/l on Day 7. After 7 days of hospitalization, the patient’s condition was stable, with no fever nor respiratory symptoms. She was discharged from the hospital and sent back to the COVID-19 isolation Center. A control of the SARS-CoV-2 RT-PCR was negative on Day 13, she returned back home. The late postpartum up to 6 weeks was unremarkable, with no complication. No medical staff involved in this case was subsequently found to be infected with SARS-CoV-2. Discussion One year after the first cases of COVID-19, the disease continues spreading at striking speed in many countries worldwide. People get contaminated mainly through direct means (including respiratory droplets and physical contacts with carriers) or by indirect contacts with contaminated objects [16]. Vertical transmission of SARS-CoV-2 during pregnancy is another possible route of transmission [9], although further investigations are still needed to confirm this eventuality. In previously published case reports of neonatal SARS-CoV-2 infections, it was not well established if the contamination occurred during pregnancy or after birth, especially during the delivery process [17]. Peritoneal lesions associated with SARS CoV-2 were previously suspected in patients [18–20]. Recently, the transplacental transmission of SARS-CoV-2 was reported [21, 22] and a classification of the COVID-19 infection in pregnant women, foetuses and newborns was suggested [23]. This case-report highlights a number of facts which suggest a possible intrauterine transmission of SARS-CoV-2 infection. We recognize, however, the limitations of our means of exploration which would confirm the case. Furthermore, none of the medical staff involved was subsequently found to be infected by SARS-CoV-2. The intense inflammatory reaction of the uterus and foetal appendages suggest a direct effect of SARS-CoV-2 on placenta. Conclusion This case is one of the first documented cases of COVID-19 in pregnancy in sub-Saharan Africa. The intense inflammatory reaction of the uterus and foetal appendages suggest a direct effect of SARS-CoV-2 on placenta. We strongly suggest obstetricians to carefully examine the aspect of the peritoneum, viscera and foetal appendages in affected pregnant women. Abbreviations COVID-19Coronavirus disease 2019 DRCDemocratic Republic of the Congo HPGRBHôpital Provincial Général de Référence de Bukavu, Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements We are very grateful to all those persons who contributed to this work, especially Dr. BULAMBO ASOKA Blandine. Authors sincerely thank all the staff of Provincial referral Hospital of Bukavu for their collaboration for this case study. Authors’ contributions EKB: drafted the manuscript, reviewed the literature, followed up the patient and edited the final manuscript. GMM, SZM, PMK, EBM, DGM, MB and GBB edited and revised the manuscript. All the authors approved the final version of the manuscript. Funding This case report received no funding. Availability of data and materials Materials and data provided in this case study are available from the corresponding author on reasonable request. Ethics approval and consent to participate The publication of this case was approved by the Ethics committee of the Catholic University of Bukavu, DRC. Consent for publication Consent for publication of the clinical details and/or laboratory results was obtained from the patient. Competing interests The authors declare that they have no competing interests.	Transplacental	DrugAdministrationRoute	CC BY	33472696	19,184,030	2021-01-20
What was the dosage of drug 'CEFTRIAXONE'?	A case study of the first pregnant woman with COVID-19 in Bukavu, eastern Democratic Republic of the Congo. BACKGROUND Vertical transmission of covid-19 is possible; its risk factors are worth researching. The placental changes found in pregnant women have a definite impact on the foetus. METHODS We report a case of a 25-year-old woman, gravida 3, para 2 (2 alive children), with a history of two caesarean deliveries, who was infected by the SARS-CoV-2 during the last term of her pregnancy. She gave birth by caesarean at 34 weeks of gestation to a newborn baby also infected with SARS-CoV-2. The peri-operative observations noted several eruptive lesions in the pelvis, bleeding on contact. Microscopic examination of the foetal appendages revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels. CONCLUSIONS This case is one of the first documented cases of COVID-19 in pregnancy in sub-Saharan Africa. We strongly suggest obstetricians to carefully examine the aspect of the peritoneum, viscera and foetal appendages in affected pregnant women. Background In December 2019, first cases of severe acute respiratory syndrome (SARS) due to a new coronavirus (SARS-CoV-2) were reported from Wuhan in China. Soon after, the disease, subsequently named “the 2019 novel coronavirus disease” (COVID-19) and declared a pandemic by the World Health Organisation (WHO) [1], has resulted in over 18.9 million confirmed cases and more than 709,000 deaths worldwide [2]. The pregnant woman can be considered to be more at risk of severe form than the non pregnant woman [3]. Their fragile immunity and frequent comorbidities such as obesity, diabetes mellitus, arterial hypertension, or cardiovascular diseases may expose them at higher risks of developing severe forms of the disease [4] and to adverse pregnancy outcomes, especially during the third trimester [5]. COVID-19 causes pneumonia with acute respiratory distress syndrome (ARDS), which can compromise natural delivery, increase maternal morbidity, or even lead to maternal death [6]. Knowledge about coronavirus disease during pregnancy is still limited [4], and vertical transmission in utero is not yet well established [4, 5]. The risk of mother-to-child transmission of COVID-19 seems to be low [7–9]. Cases of perinatal transmission of COVID-19 have been described, but it is still unclear if this occurred via the transplacental or other routes during delivery [10]. Furthermore, COVID-19 may constitute a threat of premature delivery, intrauterine growth retardation, premature rupture of the membranes, in-utero foetal death or even a premature neonatal death during delivery or soon after [8]. Among pregnant women with SARS-CoV-2, preterm birth is reported 12.9% [11]. There are no established guidelines about the best timing nor the best mode of delivery in COVID-19 infected pregnant women to optimize foetal and maternal well-being [12]. A study on the association between mode of delivery and maternal and neonatal outcomes in COVID-19 patients in Spain has shown that caesarean section was associated with an increased risk for maternal clinical deterioration which remained significant after adjustment for confounders [13]. However, a few case-reports have shown a benefit of a caesarean section on the improvement of respiratory distress in severely affected patients [5, 12]. There have been only a few studies that have investigated the intraoperative findings in women undergoing caesarean delivery [14], or changes in the foetal appendages that may explain the risk of maternal-foetal transmission [15]. We report the first documented case of COVID-19 in a pregnant woman recorded in the province of South Kivu, in eastern Democratic Republic of the Congo (DRC) who gave birth by cesarean section to a premature newborn also infected by SARS-CoV-2. Her pelvic organs exhibited a particular inflammatory appearance, and fetal appendages revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels. Case presentation A 25-year-old woman, gravida 3 para 2, at 34 weeks of gestation, with no medical history of cardiovascular nor other chronic diseases, was admitted to the labour and delivery unit of the “Hôpital Provincial Général de Référence de Bukavu” (HPGRB), in South-Kivu, for preterm labour contractions in a context of COVID-19. She had an history of 2 previous caesarean sections (the first one due to a cervical dystocia and the second indicated because of the prior caesarean), her last born wasaged 16 months. Her husband, tested negative for SARS-CoV-2, was a contact person of a COVID-19 confirmed case. Three weeks before admission, she complained of fever, not responding to acetaminophen. Her obstetrician prescribed her antibiotics, anti-malaria, and anti-spasmodic drugs. Two weeks later, as fever persisted despite all these medications, a reverse transcriptase-polymerase chain reaction (RT-PCR) nasopharyngeal swabs was performed and confirmed she was infected by SARS-CoV-2. She was then admitted to the provincial COVID-19 treatment center for isolation and health care. Upon arrival to the center, her body temperature was 38.7 °C. Gynecologic examination was unremarkable. All bacteriological tests, including hemocultures and cultures of urines were negative. She received antipyretics (acetaminophen), antispasmodics trimethylphloroglucinol and antibiotics (oral azithromycin for 5 days and intravenous ceftriaxone). Two days later, she complained of hypogastric pain, like uterine contractions of low intensity. Obstetricians of the HPGRB were contacted and recommended the administration of antispasmodics intravenously in perfusion. Despite this treatments, fever and uterine contractions persisted, so intravenous dexamethasone 12 mg daily was administered for fetal pulmonary maturation, associated with a tocolysis using nifedipine for 48 h. As the frequency, intensity and duration of contractions increased, accompanied by cervical changes (dilation, effacement, softening, and movement to a more anterior position), the patient was transferred to the labour and delivery unit of the HPGRB for an optimal care. A rapid SARS-Cov-2 antigen test was performed and found to be negative. On admission at the HPGRB, the patient had a good general condition. Her temperature (36.5 °C) and blood pressure (120/60 mmHg) were normal. The uterine height was 29 cm, the fœtus was in cephalic presentation. On vaginal examination, the uterine cervix was softened, median, 5 mm long and had a 5 cm dilatation. Membranes were intact and the fœtal head was mobile. An obstetrical ultrasound confirmed the cephalic presentation and estimated the foetal weight at 1600 g. Foetal monitoring confirmed a foetal well-being, with a stable foetal cardiac rhythm around 140 beats per minute. Tocography showed two to three contractions per minute and an intensity of 50 to 60 mmHg. A diagnosis of intractable preterm labor in a COVID-19 patient with a history of iterative caesarean deliveries was made. A classic Caesarean section with a Pfannestiel incision was performed. The peritoneal cavity and uterus were found to be very inflamed. Fetal appendages as well as the bladder were strewn with eruptive, vesicular lesions bleeding on contact (Figs. 1 and 2). The amniotic fluid was opalescent. The placenta weighed 500 g and had a clot on the maternal side on less than 20% of the surface. Anatomopathological examination subsequently revealed thrombotic vasculopathy in the placenta and in the umbilical cord vessels (Figs. 3 and 4), and a diffuse hyalinization with marked angiogenesis of the villous stroma. Fig. 1 Peri-operative appearance showing several bleeding eruptive lesions on contact ( ) with the posterior surface of the uterus, on the ovaries, the left fallopian tube up to Douglas’ pouch Fig. 2 Same lesions in the right appendages of the uterus ( ) Fig. 3 Section of the placenta; in the Hematoxylin-eosin (HE) staining we noted on sections of the placenta at 100X magnification, highly vascularized villi, with vascular lesions ( ) in the form of congestion and thrombosis visualized in certain fields and large areas of stromal hyalinization Fig. 4 Cross section of the umbilical cord (Magnification: 40X, Hematoxylline-eosin staining). There is the presence of thrombus ( ) in the lumen of the umbilical vessel, Wharton’s jelly is without distinction, no inflammatory reaction About five minutes after skin incision, a female newborn weighing 1760 g was delivered with 1 and 5 min APGAR scores of 9–10. The newborn was immediately transferred to the neonatal ward for specialized neonatal treatment for an optimal care and to minimise the potential risk of infection. Gestational age was estimated at 33 weeks according to the Finnstrom score. The newborn received the usual care (drying, stimulation, vitamin K1, Fluorescein and care of the umbilical cord). A gastric liquid was collected by gastric tube, and different swabs (especially nasopharyngeal, ear and umbilical cord), as well as blood cultures were immediately performed for bacteriological investigations and for SARS-CoV-2 RT-PCR test. The newborn was breathing autonomously, had a good control of body temperature and blood sugar. She received a 10% glucose infusion for 48 h, and on the second day an enteral feeding by nasogastric tube was progressively introduced, using artificial milk formulas adapted to preterm babies. Prophylactic antibiotherapy (penicillin G and amikacin) was initiated, considering the risk of neonatal infections in prematurity. On postnatal day 3, the newborn baby presented jaundice, respiratory distress and a clinical picture of ulcerative enterocolitis. Hemocultures were found negative, but SARS-CoV-2 RT-PCR was positive in oropharyngeal swab and cultures of gastric liquid isolated multiresistant Citrobacter sp. and Enterobacter cloacae. A phototherapy was prescribed for 3 days and previous antibiotics were replaced by meropenem and vancomycin based on the antibiogram. Despite this treatment, the newborn died on Day 5 in a picture of severe neonatal sepsis. The postoperative follow-up of the mother was marked by a persistence of fever for 3 days, varying between 39 and 40 °C. Although haemocultures and urine cultures were sterile, antibiotic therapy was readjusted on postoperative Day 3 as for the newborn, with ceftriaxone replaced by meropenem. C-reactive protein (CRP) varied from 106.53 mg/l on admission to 186 mg/l on postoperative Day 1, falling to 21.93 mg/l on Day 5 and below 3 mg/l on Day 7. After 7 days of hospitalization, the patient’s condition was stable, with no fever nor respiratory symptoms. She was discharged from the hospital and sent back to the COVID-19 isolation Center. A control of the SARS-CoV-2 RT-PCR was negative on Day 13, she returned back home. The late postpartum up to 6 weeks was unremarkable, with no complication. No medical staff involved in this case was subsequently found to be infected with SARS-CoV-2. Discussion One year after the first cases of COVID-19, the disease continues spreading at striking speed in many countries worldwide. People get contaminated mainly through direct means (including respiratory droplets and physical contacts with carriers) or by indirect contacts with contaminated objects [16]. Vertical transmission of SARS-CoV-2 during pregnancy is another possible route of transmission [9], although further investigations are still needed to confirm this eventuality. In previously published case reports of neonatal SARS-CoV-2 infections, it was not well established if the contamination occurred during pregnancy or after birth, especially during the delivery process [17]. Peritoneal lesions associated with SARS CoV-2 were previously suspected in patients [18–20]. Recently, the transplacental transmission of SARS-CoV-2 was reported [21, 22] and a classification of the COVID-19 infection in pregnant women, foetuses and newborns was suggested [23]. This case-report highlights a number of facts which suggest a possible intrauterine transmission of SARS-CoV-2 infection. We recognize, however, the limitations of our means of exploration which would confirm the case. Furthermore, none of the medical staff involved was subsequently found to be infected by SARS-CoV-2. The intense inflammatory reaction of the uterus and foetal appendages suggest a direct effect of SARS-CoV-2 on placenta. Conclusion This case is one of the first documented cases of COVID-19 in pregnancy in sub-Saharan Africa. The intense inflammatory reaction of the uterus and foetal appendages suggest a direct effect of SARS-CoV-2 on placenta. We strongly suggest obstetricians to carefully examine the aspect of the peritoneum, viscera and foetal appendages in affected pregnant women. Abbreviations COVID-19Coronavirus disease 2019 DRCDemocratic Republic of the Congo HPGRBHôpital Provincial Général de Référence de Bukavu, Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements We are very grateful to all those persons who contributed to this work, especially Dr. BULAMBO ASOKA Blandine. Authors sincerely thank all the staff of Provincial referral Hospital of Bukavu for their collaboration for this case study. Authors’ contributions EKB: drafted the manuscript, reviewed the literature, followed up the patient and edited the final manuscript. GMM, SZM, PMK, EBM, DGM, MB and GBB edited and revised the manuscript. All the authors approved the final version of the manuscript. Funding This case report received no funding. Availability of data and materials Materials and data provided in this case study are available from the corresponding author on reasonable request. Ethics approval and consent to participate The publication of this case was approved by the Ethics committee of the Catholic University of Bukavu, DRC. Consent for publication Consent for publication of the clinical details and/or laboratory results was obtained from the patient. Competing interests The authors declare that they have no competing interests.	5 DAYS	DrugDosageText	CC BY	33472696	19,183,964	2021-01-20
What was the administration route of drug 'AMPHOTERICIN B'?	Tisagenlecleucel infusion in patients with relapsed/refractory ALL and concurrent serious infection. Tisagenlecleucel, an anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy, has demonstrated durable efficacy and a manageable safety profile in pediatric and young adult patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) in the ELIANA pivotal trial and real-world experience. Experience from investigator-led studies prior to ELIANA suggests that infections and inflammatory conditions may exacerbate the severity of cytokine release syndrome (CRS) associated with CAR-T cell therapy, leading to extreme caution and strong restrictions for on-study and commercial infusion of tisagenlecleucel in patients with active infection. CRS intervention with interleukin (IL)-6 blockade and/or steroid therapy was introduced late in the course during clinical trials due to concern for potential negative effect on efficacy and persistence. However, earlier CRS intervention is now viewed more favorably. Earlier intervention and consistency in management between providers may promote broader use of tisagenlecleucel, including potential curative therapy in patients who require remission and recovery of hematopoiesis for management of severe infection. Patient 1 was diagnosed with B-ALL at 23 years old. Fourteen days before tisagenlecleucel infusion, the patient developed fever and neutropenia and was diagnosed with invasive Mucorales infection and BK virus hemorrhagic cystitis. Aggressive measures were instituted to control infection and to manage prolonged cytopenias during CAR-T cell manufacturing. Adverse events, including CRS, were manageable despite elevated inflammatory markers and active infection. The patient attained remission and recovered hematopoiesis, and infections resolved. The patient remains in remission ≥1 year postinfusion.Patient 2 was diagnosed with pre-B-ALL at preschool age. She developed severe septic shock 3 days postinitiation of lymphodepleting chemotherapy. After receiving tisagenlecleucel, she experienced CRS with cardiac dysfunction and extensive lymphadenopathy leading to renovascular compromise. The patient attained remission and was discharged in good condition to her country of origin. She remained in remission but expired on day 208 postinfusion due to cardiac arrest of unclear etiology. Infusion was feasible, and toxicity related to tisagenlecleucel was manageable despite active infections and concurrent inflammation, allowing attainment of remission in otherwise refractory pediatric/young adult ALL. This may lead to consideration of tisagenlecleucel as a potential curative therapy in patients with managed active infections. pmcBackground Anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy (tisagenlecleucel) has demonstrated durable efficacy and manageable safety for pediatric and young adult patients with relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL).1–3 In the single-arm, phase 2 ELIANA study (NCT02435849), 79 patients with r/r B-ALL (age range, 3–24 years) were infused with a single dose of tisagenlecleucel. The overall remission rate (complete remission and complete remission with incomplete blood count recovery) was 82% within 3 months. Adverse events of special interest included cytokine release syndrome (CRS, 77%) and neurological toxicities (39%).3 Patients with active infections were not enrolled and infusion was held for cases of uncontrolled infection or inflammation. Current US prescribing information warns against administration of tisagenlecleucel to patients with active infection or inflammatory conditions.1 However, there remains a lack of data for CAR-T cell therapy in patients with active, managed infections. Furthermore, there are varying levels of comfort in treating patients with different levels of disease burden, infection types, and comorbidities. The publication of clinician and patient experiences with difficult cases should benefit providers treating patients in need of potentially curative therapy but who may be delayed or denied therapy based on prior guidelines for infusion. Herein, we describe two patients with r/r B-ALL and concurrent infection and/or organ dysfunction who were safely infused with and responded to tisagenlecleucel. Case presentation Patient 1 A 23-year-old male was diagnosed with B-ALL 18 months before tisagenlecleucel infusion. His disease relapsed after standard and salvage chemotherapy with development of refractory disease following haploidentical hematopoietic stem cell transplant. An additional text file includes further treatment history (see online supplemental file). The patient was evaluated for tisagenlecleucel and leukapheresis occurred 17 months after diagnosis. Bone marrow evaluation after bridging chemotherapy revealed 95% blasts in a normocellular marrow. 10.1136/jitc-2020-001225.supp1 Supplementary data Fourteen days preinfusion, the patient was admitted for fever/neutropenia. Due to persistent fevers, a CT scan was obtained, which demonstrated findings concerning for fungal infection. He underwent right lung thoracotomy and diaphragm biopsy and was diagnosed with invasive Mucorales infection with involvement of the right lower lung lobe, diaphragm, and liver (figure 1A). Therapy was initiated with intravenous liposomal amphotericin B, micafungin, and posaconazole (switched to isavuconazonium sulfate after 4 days); intrathoracic amphotericin B deoxycholate and granulocyte infusions were also administered. The patient remained febrile with severe pancytopenia and severe concurrent BK virus hemorrhagic cystitis. He underwent thorascopic resection of the necrotic lung by wedge resection. A posterior right mini-thoracotomy was also performed for achievement of liver debridement. Following extensive discussion regarding risk of mortality due to underlying disease progression, extensive comorbid infection, and inability to attain effective immune reconstitution in the face of heavy leukemic burden in the marrow, the decision was made to proceed with tisagenlecleucel. Lymphodepleting chemotherapy (fludarabine (dose: 30 mg/m2/day) and cytarabine (dose: 1 g/m2/day), modified due to severe lymphocytopenia and known alkylator-resistant disease) was administered over 2 days. At the time of infusion, the patient was normotensive, though still febrile and hypoxic (on supplemental nasal cannula) with three chest tubes in place following thoracotomy. The patient tolerated tisagenlecleucel infusion. Figure 1 Contrast-enhanced CT scans for both patients before tisagenlecleucel and after tisagenlecleucel infusion. (A) Patient 1: CT scans performed at diagnosis and following treatment. Axial (top left) and sagittal (bottom left) images at diagnosis demonstrate a lenticular hypodense structure (yellow arrows) centered at, and Inseparable from, the abnormally thickened right diaphragm (dashed arrows). This causes invasion or indentation of the adjacent posterior right hepatic contour. A small tubular air-filled component is present within the diaphragm (red arrows). Subtle asymmetric enlargement of the right M raises the question of chest wall involvement. There is consolidation of the adjacent right lung (). Post-treatment axial (top right) and sagittal (bottom right) images demonstrate resolution of the hypodense structure, with a residual thick-walled enhancing fluid collection along the diaphragm (yellow arrows). Note improved contour of the posterior right liver. Anastomotic sutures (red arrow) and a small left pleural effusion () are present. (B) Patient 2: CT scans performed at diagnosis and following treatment. Coronal (top left) and sagittal (bottom left) images prior to treatment demonstrate mildly enhancing soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A small amount of free fluid (*) is present. Post-treatment coronal (top right) and sagittal (bottom right) images demonstrate enlargement of the soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A, aorta; I, intrahepatic inferior vena cava; M, paraspinal muscle. Fever frequency and intensity increased several days after infusion, consistent with CRS. The patient remained normotensive with minimal oxygen requirement. On postinfusion day 8, tocilizumab was administered for a temperature of 42.9°C. Fever returned 12 hours later but eventually resolved 7 days after tocilizumab administration (figure 2A). No other signs or symptoms of CRS materialized. To help decide between palliative therapy and continued care with curative intent, an early response evaluation was performed 13 days after infusion. Bone marrow obtained at that time showed acellular morphology with 0.02% leukemia by flow cytometry. Given the low leukemic burden in the bone marrow and anticipated low likelihood of recurrent CRS, attention was turned to supporting hematopoiesis, which would be required to fully eradicate the invasive fungal infection. With this, granulocyte-macrophage colony-stimulating factor (GM-CSF) was initiated and granulocyte infusions were resumed. A repeat marrow was performed on day 28 postinfusion and showed a hypocellular marrow with no immunophenotypic evidence of disease. Due to persistent cytopenias and marrow hypocellularity, consideration was given to a CD34+ stem cell-selected boost from the patient’s mother (stem cell donor); by day+34 post-infusion, however, the patient achieved a spontaneous rise in his absolute neutrophil count. Granulocyte infusions were discontinued, and stem cell donation was canceled. The patient was discharged from the hospital 39 days after infusion. He remained on isavuconazonium, liposomal amphotericin B, and GM-CSF at time of discharge. Fifty days after infusion, GM-CSF was discontinued (figure 3A); no significant side effects were observed due to the use of GM-CSF. Figure 2 Temperature, treatment, ferritin levels, and CRP levels before and after tisagenlecleucel infusion. (A) Patient 1 temperature, treatment, ferritin levels, and CRP levels. (B) Patient 2 temperature, treatment, ferritin levels, and CRP levels. CRRT, continuous renal replacement therapy; CRP, C reactive protein; Cy, cyclophosphamide; ECMO, extracorporeal membrane oxygenation; Flu, fludarabine; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; LDC, lymphodepleting chemotherapy. Figure 3 Timeline of infections and treatment throughout the tisagenlecleucel therapy process. (A) Timeline for patient 1. aDay 8 of cefepime found muscle swelling and fungal elements in the liver; thus, the patient underwent surgery (right video-assisted thorascopic surgery with right lower lobe lung wedge resection, right thoracotomy, diaphragm biopsy, and right liver lobe wedge resection). bDiscontinued 2 days before infusion and restarted 14 days after until end of September 2018. cVideo-assisted thorascopic surgery in which a small portion of the right middle lung lobe was resected from the pleura and showed negative fungal elements. Lesion was not patched due to the potential of fungal growth. dOngoing observation. (B) Timeline for patient 2. aDelayed to December 31 due to infection. bPositive for rhinovirus/enterovirus, adenovirus, and BK virus, but remained asymptomatic. cContinued throughout blast count recovery. CRRT, continuous renal replacement therapy; GM-CSF, granulocyte-macrophage colony-stimulating factor; IVIG, intravenous immunoglobulin; LD, lymphodepleting; VA-ECMO, venoarterial extracorporeal membrane oxygenation. Forty-nine days after infusion, an additional thoracoscopic surgery was performed to resect residual necrotic lung; no fungal elements were detected at that time (figure 1A), and antifungal therapy was narrowed to monotherapy with isavuconazonium. The patient returned to his country of origin 76 days after infusion and remains in remission 19 months after infusion. Patient 2 A preschool-age Hispanic female was diagnosed with pre-B-ALL 14 months before tisagenlecleucel infusion and was treated according to the National Mexican Protocol, initially with achievement of complete remission (see online supplemental file). She developed relapsed disease 7 months prior to tisagenlecleucel infusion. She underwent reinduction chemotherapy, blinatumomab, and focal radiation; despite these therapies, bone marrow evaluation continued to show active disease 1 year after diagnosis, at which time she was referred for tisagenlecleucel infusion. The patient underwent leukapheresis 1 month prior to tisagenlecleucel infusion. She received bridging chemotherapy of 6-mercaptopurine and methotrexate and underwent lymphodepleting chemotherapy of fludarabine (30 mg/m2/dose for 3 days) and cyclophosphamide (500 mg/m2/dose for 2 days) 18 days prior to tisagenlecleucel infusion. Within 3 days of starting lymphodepleting chemotherapy, while neutropenic, she developed severe septic shock (extended spectrum beta-lactamase-positive and ertapenem-resistant Escherichia coli) with need for broad-spectrum antibiotics, steroids, inotropes, ventilatory support, 4 days of continuous renal replacement therapy and 5 days of venoarterial extracorporeal membrane oxygenation (VA-ECMO). VA-ECMO was discontinued 11 days prior to tisagenlecleucel infusion. Shortly thereafter, the patient developed recurrent fever. A CT scan obtained for evaluation of an infectious nidus showed pulmonary opacities with tree-in-bud appearance (possible fungal etiology) and a small intra-abdominal abscess/phlegmon. Given these findings, she was started on posaconazole in addition to existing broad-spectrum antimicrobials (figure 2B). With consideration given to disease burden (11% circulating leukemic blasts on day prior to infusion), optimal antifungal/antimicrobial therapy, and stable clinical exam, the decision was made to proceed with tisagenlecleucel infusion (figure 1B). At the time of infusion, respiratory failure was resolved (without need for non-invasive ventilatory support) and the patient was hemodynamically stable. She remained on amlodipine for management of underlying hypertension, but all inotropic support was discontinued. An echocardiogram (obtained 4 days preinfusion) showed qualitatively normal right ventricular systolic function and mild global hypokinesis of the left ventricle (ejection fraction (EF) 49%, shortening fraction (SF) 32%). The patient’s postinfusion course was complicated by CRS (characterized by fever, cardiac dysfunction, fluid overload, and respiratory insufficiency) and extensive lymphadenopathy (figure 1B) with associated renovascular compromise. She received two doses of tocilizumab on day+21 given 12 hours apart with some clinical improvement, though ultimately with need for resumption of non-invasive ventilatory support and milrinone due to depressed left ventricular systolic function (EF 45%, SF 21.5%). Given the unclear nature of the patient’s aforementioned extensive adenopathy and inability to exclude disease progression, a bone marrow biopsy and aspirate were completed on day 21 postinfusion; it revealed persistent marrow disease, though notably with numerous CD3+ T lymphocytes surrounding visualized blast clusters (figure 4A–E). Follow-up biopsy and aspirate obtained 31 days postinfusion showed extensive fibrosis with no morphological or immunophenotypic evidence of disease (figure 4F). The patient was discharged from the hospital 53 days after tisagenlecleucel infusion. An echocardiogram obtained prior to discharge showed mildly depressed left ventricle systolic function (EF 49%, SF 26%) with mild left ventricular dilation. At the time of discharge (day+54 postinfusion) and on transfer home to her country of origin (day+58), she remained on amlodipine for management of hypertension but was otherwise on no cardiac remodeling medications. She remained on granulocyte colony-stimulating factor for management of severe neutropenia and was transfusion-dependent (packed red blood cells and platelets). In the setting of severe neutropenia, levofloxacin and voriconazole were also continued for infection prophylaxis. Figure 4 Bone marrow biopsy for patient 2 before and after tisagenlecleucel. (A–F) Pathology of biopsies for patient 2. Pathology of the bone marrow at 21 days (A–E) and 31 days (F) after infusion. (A, B) Blasts in the bone marrow exhibited karyorrhexis (blue arrows) mixed with intact peripheral cells 21 days after infusion. (C) CD3+ T cells surrounding and infiltrating leukemic blasts. Blue arrows indicate CD3-negative blast cells. (D) CD19 expression on the blasts. (E) Dual CD3-CD10+ immunostaining highlight T cells in brown (black arrows) and blasts in red (blue arrows). (F) Bone marrow at day 31, depleted of hematopoietic cells. The patient returned to her country of origin 58 days after infusion. Unfortunately, she expired 208 days after infusion due to cardiac arrest of unclear etiology (figure 3B). An autopsy was not obtained. Her last bone marrow evaluation was completed 3 weeks prior to death and showed no morphological or immunophenotypic evidence of disease. Discussion Here, we describe two patients with heavily pretreated r/r B-ALL who were safely infused with tisagenlecleucel despite elevated inflammatory markers, heavy leukemic burden, significant infection, and multiple comorbidities. While our center has experience with infusion of CAR-T cells dating back to 2012 and physician experience with infusions dating to 2005, tisagenlecleucel infusions are and will be the first such infusions for many centers. The complex management of patients prior to, during, and after infusion can be a source of significant concern for many providers, particularly in the presence of underlying infection, evolving disease burden, and/or other confounding variables that increase risk for severe CRS. Early recommendations to sites regarding conditions for infusion and CRS management on the pivotal multisite ELIANA trial of tisagenlecleucel were based on adult and pediatric experience at a single institution. During the trial, experienced providers were frequently consulted by those centers or providers with less experience in CAR-T cell infusion when challenged by cases for which the existing CRS management guidelines did not provide clear guidance on how to proceed (ie, a provider may inquire about tocilizumab for a patient with 5 days of persistent fever to 40°C without other signs or symptoms of CRS). Earlier in the ELIANA trial, there was significant concern that early CRS intervention might lead to diminished efficacy. Through increased patient experience in both trial and real-world settings, however, much has been learned about patient management, including management of mild to severe CRS and neurological toxicity.4 As the number of sites administering tisagenlecleucel expands and a wide range of real-world experience is obtained, it will be difficult to implement rapid changes to official guidelines. In the ELIANA trial, 43% of patients experienced infections within 8 weeks of tisagenlecleucel infusion, and 24% were severe infections (grade 3/4).2 In a few patients, prolonged severe neutropenia led to severe (grade 3 human herpesvirus 6 encephalitis) and fatal (encephalitis and systemic mycosis) infections. Analysis of real-world data in patients with B-ALL from the Center for International Blood and Marrow Transplant Research (CIBMTR) patient registry reported a similar incidence of postinfusion infections (42%), with one fatal bacterial infection.5 Furthermore, management of CRS with short-term tocilizumab therapy (IL-6 blockade) does not appear to increase the risk of infection6; however, it is important to consider that immune responses to invasive fungal infections could involve IL-67 8 and therefore may respond differently to tocilizumab therapy than was reported for our patient. Taken together, these clinical trial and real-world data highlight the known postinfusion infection risk in these patients; however, existing data fail to provide insight into how patients with preinfusion infections might be managed. Expanding the existing literature to further describe management of complex patients who, in some respects, challenge the current guidelines for infusion will be important for improving access to therapy for patients who lack other options for potential durable remission. Technically, the patients reported here were infused with tisagenlecleucel outside of label guidelines due to the presence of elevated inflammatory markers and active infection.1 For patient 1, CRS manifestations were limited to only fever without hypotension or progressive renal or pulmonary involvement. Response of B-ALL to tisagenlecleucel was not compromised by the treatment of CRS at this stage with tocilizumab. For patient 2, CRS was characterized by fever, third-spacing, cardiac dysfunction, and marked lymphadenopathy with secondary renal vascular compromise (suspected to be driven by CAR-T cell expansion). Cardiac dysfunction due to CRS has been observed in children undergoing CAR-T cell therapy, and risk of dysfunction may be increased in patients who have pre-existing cardiac abnormalities; persistent dysfunction remains uncommon, however.9 10 For patient 2, improvement in cardiac function was observed following resolution of CRS, though with unexpected death due to cardiac arrest greater than 200 days after infusion. The etiology of this arrest remains unknown at this time. Through experience gained treating CRS in patients who were part of the ELIANA study, we felt relatively comfortable that we could control CRS and therefore infused both patients under circumstances in which other providers might not have. Nevertheless, tisagenlecleucel infusion was only carried out after extensive counseling regarding the possibility of worst-case scenarios and with serious consideration also given to transport home for palliative measures. In both cases, significant discussions regarding potential life-threatening outcomes were had with patients, families, and medical providers prior to infusion. Conclusions Publication of these case studies and others may guide providers and families as they face similarly challenging circumstances in the future. In addition, they highlight the importance of clear communication with patients, families, and medical team members about potentially lethal adverse events of CAR-T cell therapy when infused in the setting of concurrent life-threatening comorbidities. While these cases represent successful infection management during CAR-T cell therapy, they should not be viewed as typical or even likely outcomes. Treatment of both patients was carried out in a setting with extensive institutional knowledge by an experienced team with the ability to customize standard protocols. Additional reporting of outcomes in patients with active infections via the CIBMTR patient registry is needed to confirm the generalizability of our approach and to better delineate benefit–risk assessment in these patients. Careful reporting on patient experiences and outcomes in the postcommercialization era of tisagenlecleucel will help to ensure safe access to potentially curative therapy for more patients. Ethics statements Patient consent for publication Obtained. Ethics approval This is a retrospective case series for which institutional review board permissions were granted and results were pulled from patients’ charts. Correction notice: This article has been corrected since it first published. The provenance and peer review statement has been included. Contributors: All authors treated patients and contributed to data collection and interpretation. EMH and GDM provided data and helped write the first draft. All authors were involved in revising the manuscript, and read and approved the final version. Funding: Medical writing support was provided by Briana Dye, PhD, of Healthcare Consultancy Group, and was funded by Novartis Pharmaceuticals Corporation. Competing interests: DEY has been an investigator on studies supported by Astellas, Chimerix, Merck, Pfizer, and Viracor-Eurofins. KJA is on the Speaker’s Bureau for and receives support from Novartis Pharmaceuticals. GDM is a consultant and is on the Speaker’s Bureau for Novartis Pharmaceuticals. EMH, RKG, AAA, GSM, SDS, TGF, IAA, WL, and RJH declare that they have no competing interests. Provenance and peer review: Not commissioned; externally peer reviewed. Supplemental material: This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.	Intravenous (not otherwise specified)	DrugAdministrationRoute	CC BY-NC	33472856	19,003,409	2021-01
What was the administration route of drug 'TISAGENLECLEUCEL'?	Tisagenlecleucel infusion in patients with relapsed/refractory ALL and concurrent serious infection. Tisagenlecleucel, an anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy, has demonstrated durable efficacy and a manageable safety profile in pediatric and young adult patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) in the ELIANA pivotal trial and real-world experience. Experience from investigator-led studies prior to ELIANA suggests that infections and inflammatory conditions may exacerbate the severity of cytokine release syndrome (CRS) associated with CAR-T cell therapy, leading to extreme caution and strong restrictions for on-study and commercial infusion of tisagenlecleucel in patients with active infection. CRS intervention with interleukin (IL)-6 blockade and/or steroid therapy was introduced late in the course during clinical trials due to concern for potential negative effect on efficacy and persistence. However, earlier CRS intervention is now viewed more favorably. Earlier intervention and consistency in management between providers may promote broader use of tisagenlecleucel, including potential curative therapy in patients who require remission and recovery of hematopoiesis for management of severe infection. Patient 1 was diagnosed with B-ALL at 23 years old. Fourteen days before tisagenlecleucel infusion, the patient developed fever and neutropenia and was diagnosed with invasive Mucorales infection and BK virus hemorrhagic cystitis. Aggressive measures were instituted to control infection and to manage prolonged cytopenias during CAR-T cell manufacturing. Adverse events, including CRS, were manageable despite elevated inflammatory markers and active infection. The patient attained remission and recovered hematopoiesis, and infections resolved. The patient remains in remission ≥1 year postinfusion.Patient 2 was diagnosed with pre-B-ALL at preschool age. She developed severe septic shock 3 days postinitiation of lymphodepleting chemotherapy. After receiving tisagenlecleucel, she experienced CRS with cardiac dysfunction and extensive lymphadenopathy leading to renovascular compromise. The patient attained remission and was discharged in good condition to her country of origin. She remained in remission but expired on day 208 postinfusion due to cardiac arrest of unclear etiology. Infusion was feasible, and toxicity related to tisagenlecleucel was manageable despite active infections and concurrent inflammation, allowing attainment of remission in otherwise refractory pediatric/young adult ALL. This may lead to consideration of tisagenlecleucel as a potential curative therapy in patients with managed active infections. pmcBackground Anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy (tisagenlecleucel) has demonstrated durable efficacy and manageable safety for pediatric and young adult patients with relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL).1–3 In the single-arm, phase 2 ELIANA study (NCT02435849), 79 patients with r/r B-ALL (age range, 3–24 years) were infused with a single dose of tisagenlecleucel. The overall remission rate (complete remission and complete remission with incomplete blood count recovery) was 82% within 3 months. Adverse events of special interest included cytokine release syndrome (CRS, 77%) and neurological toxicities (39%).3 Patients with active infections were not enrolled and infusion was held for cases of uncontrolled infection or inflammation. Current US prescribing information warns against administration of tisagenlecleucel to patients with active infection or inflammatory conditions.1 However, there remains a lack of data for CAR-T cell therapy in patients with active, managed infections. Furthermore, there are varying levels of comfort in treating patients with different levels of disease burden, infection types, and comorbidities. The publication of clinician and patient experiences with difficult cases should benefit providers treating patients in need of potentially curative therapy but who may be delayed or denied therapy based on prior guidelines for infusion. Herein, we describe two patients with r/r B-ALL and concurrent infection and/or organ dysfunction who were safely infused with and responded to tisagenlecleucel. Case presentation Patient 1 A 23-year-old male was diagnosed with B-ALL 18 months before tisagenlecleucel infusion. His disease relapsed after standard and salvage chemotherapy with development of refractory disease following haploidentical hematopoietic stem cell transplant. An additional text file includes further treatment history (see online supplemental file). The patient was evaluated for tisagenlecleucel and leukapheresis occurred 17 months after diagnosis. Bone marrow evaluation after bridging chemotherapy revealed 95% blasts in a normocellular marrow. 10.1136/jitc-2020-001225.supp1 Supplementary data Fourteen days preinfusion, the patient was admitted for fever/neutropenia. Due to persistent fevers, a CT scan was obtained, which demonstrated findings concerning for fungal infection. He underwent right lung thoracotomy and diaphragm biopsy and was diagnosed with invasive Mucorales infection with involvement of the right lower lung lobe, diaphragm, and liver (figure 1A). Therapy was initiated with intravenous liposomal amphotericin B, micafungin, and posaconazole (switched to isavuconazonium sulfate after 4 days); intrathoracic amphotericin B deoxycholate and granulocyte infusions were also administered. The patient remained febrile with severe pancytopenia and severe concurrent BK virus hemorrhagic cystitis. He underwent thorascopic resection of the necrotic lung by wedge resection. A posterior right mini-thoracotomy was also performed for achievement of liver debridement. Following extensive discussion regarding risk of mortality due to underlying disease progression, extensive comorbid infection, and inability to attain effective immune reconstitution in the face of heavy leukemic burden in the marrow, the decision was made to proceed with tisagenlecleucel. Lymphodepleting chemotherapy (fludarabine (dose: 30 mg/m2/day) and cytarabine (dose: 1 g/m2/day), modified due to severe lymphocytopenia and known alkylator-resistant disease) was administered over 2 days. At the time of infusion, the patient was normotensive, though still febrile and hypoxic (on supplemental nasal cannula) with three chest tubes in place following thoracotomy. The patient tolerated tisagenlecleucel infusion. Figure 1 Contrast-enhanced CT scans for both patients before tisagenlecleucel and after tisagenlecleucel infusion. (A) Patient 1: CT scans performed at diagnosis and following treatment. Axial (top left) and sagittal (bottom left) images at diagnosis demonstrate a lenticular hypodense structure (yellow arrows) centered at, and Inseparable from, the abnormally thickened right diaphragm (dashed arrows). This causes invasion or indentation of the adjacent posterior right hepatic contour. A small tubular air-filled component is present within the diaphragm (red arrows). Subtle asymmetric enlargement of the right M raises the question of chest wall involvement. There is consolidation of the adjacent right lung (). Post-treatment axial (top right) and sagittal (bottom right) images demonstrate resolution of the hypodense structure, with a residual thick-walled enhancing fluid collection along the diaphragm (yellow arrows). Note improved contour of the posterior right liver. Anastomotic sutures (red arrow) and a small left pleural effusion () are present. (B) Patient 2: CT scans performed at diagnosis and following treatment. Coronal (top left) and sagittal (bottom left) images prior to treatment demonstrate mildly enhancing soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A small amount of free fluid (*) is present. Post-treatment coronal (top right) and sagittal (bottom right) images demonstrate enlargement of the soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A, aorta; I, intrahepatic inferior vena cava; M, paraspinal muscle. Fever frequency and intensity increased several days after infusion, consistent with CRS. The patient remained normotensive with minimal oxygen requirement. On postinfusion day 8, tocilizumab was administered for a temperature of 42.9°C. Fever returned 12 hours later but eventually resolved 7 days after tocilizumab administration (figure 2A). No other signs or symptoms of CRS materialized. To help decide between palliative therapy and continued care with curative intent, an early response evaluation was performed 13 days after infusion. Bone marrow obtained at that time showed acellular morphology with 0.02% leukemia by flow cytometry. Given the low leukemic burden in the bone marrow and anticipated low likelihood of recurrent CRS, attention was turned to supporting hematopoiesis, which would be required to fully eradicate the invasive fungal infection. With this, granulocyte-macrophage colony-stimulating factor (GM-CSF) was initiated and granulocyte infusions were resumed. A repeat marrow was performed on day 28 postinfusion and showed a hypocellular marrow with no immunophenotypic evidence of disease. Due to persistent cytopenias and marrow hypocellularity, consideration was given to a CD34+ stem cell-selected boost from the patient’s mother (stem cell donor); by day+34 post-infusion, however, the patient achieved a spontaneous rise in his absolute neutrophil count. Granulocyte infusions were discontinued, and stem cell donation was canceled. The patient was discharged from the hospital 39 days after infusion. He remained on isavuconazonium, liposomal amphotericin B, and GM-CSF at time of discharge. Fifty days after infusion, GM-CSF was discontinued (figure 3A); no significant side effects were observed due to the use of GM-CSF. Figure 2 Temperature, treatment, ferritin levels, and CRP levels before and after tisagenlecleucel infusion. (A) Patient 1 temperature, treatment, ferritin levels, and CRP levels. (B) Patient 2 temperature, treatment, ferritin levels, and CRP levels. CRRT, continuous renal replacement therapy; CRP, C reactive protein; Cy, cyclophosphamide; ECMO, extracorporeal membrane oxygenation; Flu, fludarabine; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; LDC, lymphodepleting chemotherapy. Figure 3 Timeline of infections and treatment throughout the tisagenlecleucel therapy process. (A) Timeline for patient 1. aDay 8 of cefepime found muscle swelling and fungal elements in the liver; thus, the patient underwent surgery (right video-assisted thorascopic surgery with right lower lobe lung wedge resection, right thoracotomy, diaphragm biopsy, and right liver lobe wedge resection). bDiscontinued 2 days before infusion and restarted 14 days after until end of September 2018. cVideo-assisted thorascopic surgery in which a small portion of the right middle lung lobe was resected from the pleura and showed negative fungal elements. Lesion was not patched due to the potential of fungal growth. dOngoing observation. (B) Timeline for patient 2. aDelayed to December 31 due to infection. bPositive for rhinovirus/enterovirus, adenovirus, and BK virus, but remained asymptomatic. cContinued throughout blast count recovery. CRRT, continuous renal replacement therapy; GM-CSF, granulocyte-macrophage colony-stimulating factor; IVIG, intravenous immunoglobulin; LD, lymphodepleting; VA-ECMO, venoarterial extracorporeal membrane oxygenation. Forty-nine days after infusion, an additional thoracoscopic surgery was performed to resect residual necrotic lung; no fungal elements were detected at that time (figure 1A), and antifungal therapy was narrowed to monotherapy with isavuconazonium. The patient returned to his country of origin 76 days after infusion and remains in remission 19 months after infusion. Patient 2 A preschool-age Hispanic female was diagnosed with pre-B-ALL 14 months before tisagenlecleucel infusion and was treated according to the National Mexican Protocol, initially with achievement of complete remission (see online supplemental file). She developed relapsed disease 7 months prior to tisagenlecleucel infusion. She underwent reinduction chemotherapy, blinatumomab, and focal radiation; despite these therapies, bone marrow evaluation continued to show active disease 1 year after diagnosis, at which time she was referred for tisagenlecleucel infusion. The patient underwent leukapheresis 1 month prior to tisagenlecleucel infusion. She received bridging chemotherapy of 6-mercaptopurine and methotrexate and underwent lymphodepleting chemotherapy of fludarabine (30 mg/m2/dose for 3 days) and cyclophosphamide (500 mg/m2/dose for 2 days) 18 days prior to tisagenlecleucel infusion. Within 3 days of starting lymphodepleting chemotherapy, while neutropenic, she developed severe septic shock (extended spectrum beta-lactamase-positive and ertapenem-resistant Escherichia coli) with need for broad-spectrum antibiotics, steroids, inotropes, ventilatory support, 4 days of continuous renal replacement therapy and 5 days of venoarterial extracorporeal membrane oxygenation (VA-ECMO). VA-ECMO was discontinued 11 days prior to tisagenlecleucel infusion. Shortly thereafter, the patient developed recurrent fever. A CT scan obtained for evaluation of an infectious nidus showed pulmonary opacities with tree-in-bud appearance (possible fungal etiology) and a small intra-abdominal abscess/phlegmon. Given these findings, she was started on posaconazole in addition to existing broad-spectrum antimicrobials (figure 2B). With consideration given to disease burden (11% circulating leukemic blasts on day prior to infusion), optimal antifungal/antimicrobial therapy, and stable clinical exam, the decision was made to proceed with tisagenlecleucel infusion (figure 1B). At the time of infusion, respiratory failure was resolved (without need for non-invasive ventilatory support) and the patient was hemodynamically stable. She remained on amlodipine for management of underlying hypertension, but all inotropic support was discontinued. An echocardiogram (obtained 4 days preinfusion) showed qualitatively normal right ventricular systolic function and mild global hypokinesis of the left ventricle (ejection fraction (EF) 49%, shortening fraction (SF) 32%). The patient’s postinfusion course was complicated by CRS (characterized by fever, cardiac dysfunction, fluid overload, and respiratory insufficiency) and extensive lymphadenopathy (figure 1B) with associated renovascular compromise. She received two doses of tocilizumab on day+21 given 12 hours apart with some clinical improvement, though ultimately with need for resumption of non-invasive ventilatory support and milrinone due to depressed left ventricular systolic function (EF 45%, SF 21.5%). Given the unclear nature of the patient’s aforementioned extensive adenopathy and inability to exclude disease progression, a bone marrow biopsy and aspirate were completed on day 21 postinfusion; it revealed persistent marrow disease, though notably with numerous CD3+ T lymphocytes surrounding visualized blast clusters (figure 4A–E). Follow-up biopsy and aspirate obtained 31 days postinfusion showed extensive fibrosis with no morphological or immunophenotypic evidence of disease (figure 4F). The patient was discharged from the hospital 53 days after tisagenlecleucel infusion. An echocardiogram obtained prior to discharge showed mildly depressed left ventricle systolic function (EF 49%, SF 26%) with mild left ventricular dilation. At the time of discharge (day+54 postinfusion) and on transfer home to her country of origin (day+58), she remained on amlodipine for management of hypertension but was otherwise on no cardiac remodeling medications. She remained on granulocyte colony-stimulating factor for management of severe neutropenia and was transfusion-dependent (packed red blood cells and platelets). In the setting of severe neutropenia, levofloxacin and voriconazole were also continued for infection prophylaxis. Figure 4 Bone marrow biopsy for patient 2 before and after tisagenlecleucel. (A–F) Pathology of biopsies for patient 2. Pathology of the bone marrow at 21 days (A–E) and 31 days (F) after infusion. (A, B) Blasts in the bone marrow exhibited karyorrhexis (blue arrows) mixed with intact peripheral cells 21 days after infusion. (C) CD3+ T cells surrounding and infiltrating leukemic blasts. Blue arrows indicate CD3-negative blast cells. (D) CD19 expression on the blasts. (E) Dual CD3-CD10+ immunostaining highlight T cells in brown (black arrows) and blasts in red (blue arrows). (F) Bone marrow at day 31, depleted of hematopoietic cells. The patient returned to her country of origin 58 days after infusion. Unfortunately, she expired 208 days after infusion due to cardiac arrest of unclear etiology (figure 3B). An autopsy was not obtained. Her last bone marrow evaluation was completed 3 weeks prior to death and showed no morphological or immunophenotypic evidence of disease. Discussion Here, we describe two patients with heavily pretreated r/r B-ALL who were safely infused with tisagenlecleucel despite elevated inflammatory markers, heavy leukemic burden, significant infection, and multiple comorbidities. While our center has experience with infusion of CAR-T cells dating back to 2012 and physician experience with infusions dating to 2005, tisagenlecleucel infusions are and will be the first such infusions for many centers. The complex management of patients prior to, during, and after infusion can be a source of significant concern for many providers, particularly in the presence of underlying infection, evolving disease burden, and/or other confounding variables that increase risk for severe CRS. Early recommendations to sites regarding conditions for infusion and CRS management on the pivotal multisite ELIANA trial of tisagenlecleucel were based on adult and pediatric experience at a single institution. During the trial, experienced providers were frequently consulted by those centers or providers with less experience in CAR-T cell infusion when challenged by cases for which the existing CRS management guidelines did not provide clear guidance on how to proceed (ie, a provider may inquire about tocilizumab for a patient with 5 days of persistent fever to 40°C without other signs or symptoms of CRS). Earlier in the ELIANA trial, there was significant concern that early CRS intervention might lead to diminished efficacy. Through increased patient experience in both trial and real-world settings, however, much has been learned about patient management, including management of mild to severe CRS and neurological toxicity.4 As the number of sites administering tisagenlecleucel expands and a wide range of real-world experience is obtained, it will be difficult to implement rapid changes to official guidelines. In the ELIANA trial, 43% of patients experienced infections within 8 weeks of tisagenlecleucel infusion, and 24% were severe infections (grade 3/4).2 In a few patients, prolonged severe neutropenia led to severe (grade 3 human herpesvirus 6 encephalitis) and fatal (encephalitis and systemic mycosis) infections. Analysis of real-world data in patients with B-ALL from the Center for International Blood and Marrow Transplant Research (CIBMTR) patient registry reported a similar incidence of postinfusion infections (42%), with one fatal bacterial infection.5 Furthermore, management of CRS with short-term tocilizumab therapy (IL-6 blockade) does not appear to increase the risk of infection6; however, it is important to consider that immune responses to invasive fungal infections could involve IL-67 8 and therefore may respond differently to tocilizumab therapy than was reported for our patient. Taken together, these clinical trial and real-world data highlight the known postinfusion infection risk in these patients; however, existing data fail to provide insight into how patients with preinfusion infections might be managed. Expanding the existing literature to further describe management of complex patients who, in some respects, challenge the current guidelines for infusion will be important for improving access to therapy for patients who lack other options for potential durable remission. Technically, the patients reported here were infused with tisagenlecleucel outside of label guidelines due to the presence of elevated inflammatory markers and active infection.1 For patient 1, CRS manifestations were limited to only fever without hypotension or progressive renal or pulmonary involvement. Response of B-ALL to tisagenlecleucel was not compromised by the treatment of CRS at this stage with tocilizumab. For patient 2, CRS was characterized by fever, third-spacing, cardiac dysfunction, and marked lymphadenopathy with secondary renal vascular compromise (suspected to be driven by CAR-T cell expansion). Cardiac dysfunction due to CRS has been observed in children undergoing CAR-T cell therapy, and risk of dysfunction may be increased in patients who have pre-existing cardiac abnormalities; persistent dysfunction remains uncommon, however.9 10 For patient 2, improvement in cardiac function was observed following resolution of CRS, though with unexpected death due to cardiac arrest greater than 200 days after infusion. The etiology of this arrest remains unknown at this time. Through experience gained treating CRS in patients who were part of the ELIANA study, we felt relatively comfortable that we could control CRS and therefore infused both patients under circumstances in which other providers might not have. Nevertheless, tisagenlecleucel infusion was only carried out after extensive counseling regarding the possibility of worst-case scenarios and with serious consideration also given to transport home for palliative measures. In both cases, significant discussions regarding potential life-threatening outcomes were had with patients, families, and medical providers prior to infusion. Conclusions Publication of these case studies and others may guide providers and families as they face similarly challenging circumstances in the future. In addition, they highlight the importance of clear communication with patients, families, and medical team members about potentially lethal adverse events of CAR-T cell therapy when infused in the setting of concurrent life-threatening comorbidities. While these cases represent successful infection management during CAR-T cell therapy, they should not be viewed as typical or even likely outcomes. Treatment of both patients was carried out in a setting with extensive institutional knowledge by an experienced team with the ability to customize standard protocols. Additional reporting of outcomes in patients with active infections via the CIBMTR patient registry is needed to confirm the generalizability of our approach and to better delineate benefit–risk assessment in these patients. Careful reporting on patient experiences and outcomes in the postcommercialization era of tisagenlecleucel will help to ensure safe access to potentially curative therapy for more patients. Ethics statements Patient consent for publication Obtained. Ethics approval This is a retrospective case series for which institutional review board permissions were granted and results were pulled from patients’ charts. Correction notice: This article has been corrected since it first published. The provenance and peer review statement has been included. Contributors: All authors treated patients and contributed to data collection and interpretation. EMH and GDM provided data and helped write the first draft. All authors were involved in revising the manuscript, and read and approved the final version. Funding: Medical writing support was provided by Briana Dye, PhD, of Healthcare Consultancy Group, and was funded by Novartis Pharmaceuticals Corporation. Competing interests: DEY has been an investigator on studies supported by Astellas, Chimerix, Merck, Pfizer, and Viracor-Eurofins. KJA is on the Speaker’s Bureau for and receives support from Novartis Pharmaceuticals. GDM is a consultant and is on the Speaker’s Bureau for Novartis Pharmaceuticals. EMH, RKG, AAA, GSM, SDS, TGF, IAA, WL, and RJH declare that they have no competing interests. Provenance and peer review: Not commissioned; externally peer reviewed. Supplemental material: This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.	Other	DrugAdministrationRoute	CC BY-NC	33472856	19,003,489	2021-01
What was the dosage of drug 'ASPARAGINASE'?	Tisagenlecleucel infusion in patients with relapsed/refractory ALL and concurrent serious infection. Tisagenlecleucel, an anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy, has demonstrated durable efficacy and a manageable safety profile in pediatric and young adult patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) in the ELIANA pivotal trial and real-world experience. Experience from investigator-led studies prior to ELIANA suggests that infections and inflammatory conditions may exacerbate the severity of cytokine release syndrome (CRS) associated with CAR-T cell therapy, leading to extreme caution and strong restrictions for on-study and commercial infusion of tisagenlecleucel in patients with active infection. CRS intervention with interleukin (IL)-6 blockade and/or steroid therapy was introduced late in the course during clinical trials due to concern for potential negative effect on efficacy and persistence. However, earlier CRS intervention is now viewed more favorably. Earlier intervention and consistency in management between providers may promote broader use of tisagenlecleucel, including potential curative therapy in patients who require remission and recovery of hematopoiesis for management of severe infection. Patient 1 was diagnosed with B-ALL at 23 years old. Fourteen days before tisagenlecleucel infusion, the patient developed fever and neutropenia and was diagnosed with invasive Mucorales infection and BK virus hemorrhagic cystitis. Aggressive measures were instituted to control infection and to manage prolonged cytopenias during CAR-T cell manufacturing. Adverse events, including CRS, were manageable despite elevated inflammatory markers and active infection. The patient attained remission and recovered hematopoiesis, and infections resolved. The patient remains in remission ≥1 year postinfusion.Patient 2 was diagnosed with pre-B-ALL at preschool age. She developed severe septic shock 3 days postinitiation of lymphodepleting chemotherapy. After receiving tisagenlecleucel, she experienced CRS with cardiac dysfunction and extensive lymphadenopathy leading to renovascular compromise. The patient attained remission and was discharged in good condition to her country of origin. She remained in remission but expired on day 208 postinfusion due to cardiac arrest of unclear etiology. Infusion was feasible, and toxicity related to tisagenlecleucel was manageable despite active infections and concurrent inflammation, allowing attainment of remission in otherwise refractory pediatric/young adult ALL. This may lead to consideration of tisagenlecleucel as a potential curative therapy in patients with managed active infections. pmcBackground Anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy (tisagenlecleucel) has demonstrated durable efficacy and manageable safety for pediatric and young adult patients with relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL).1–3 In the single-arm, phase 2 ELIANA study (NCT02435849), 79 patients with r/r B-ALL (age range, 3–24 years) were infused with a single dose of tisagenlecleucel. The overall remission rate (complete remission and complete remission with incomplete blood count recovery) was 82% within 3 months. Adverse events of special interest included cytokine release syndrome (CRS, 77%) and neurological toxicities (39%).3 Patients with active infections were not enrolled and infusion was held for cases of uncontrolled infection or inflammation. Current US prescribing information warns against administration of tisagenlecleucel to patients with active infection or inflammatory conditions.1 However, there remains a lack of data for CAR-T cell therapy in patients with active, managed infections. Furthermore, there are varying levels of comfort in treating patients with different levels of disease burden, infection types, and comorbidities. The publication of clinician and patient experiences with difficult cases should benefit providers treating patients in need of potentially curative therapy but who may be delayed or denied therapy based on prior guidelines for infusion. Herein, we describe two patients with r/r B-ALL and concurrent infection and/or organ dysfunction who were safely infused with and responded to tisagenlecleucel. Case presentation Patient 1 A 23-year-old male was diagnosed with B-ALL 18 months before tisagenlecleucel infusion. His disease relapsed after standard and salvage chemotherapy with development of refractory disease following haploidentical hematopoietic stem cell transplant. An additional text file includes further treatment history (see online supplemental file). The patient was evaluated for tisagenlecleucel and leukapheresis occurred 17 months after diagnosis. Bone marrow evaluation after bridging chemotherapy revealed 95% blasts in a normocellular marrow. 10.1136/jitc-2020-001225.supp1 Supplementary data Fourteen days preinfusion, the patient was admitted for fever/neutropenia. Due to persistent fevers, a CT scan was obtained, which demonstrated findings concerning for fungal infection. He underwent right lung thoracotomy and diaphragm biopsy and was diagnosed with invasive Mucorales infection with involvement of the right lower lung lobe, diaphragm, and liver (figure 1A). Therapy was initiated with intravenous liposomal amphotericin B, micafungin, and posaconazole (switched to isavuconazonium sulfate after 4 days); intrathoracic amphotericin B deoxycholate and granulocyte infusions were also administered. The patient remained febrile with severe pancytopenia and severe concurrent BK virus hemorrhagic cystitis. He underwent thorascopic resection of the necrotic lung by wedge resection. A posterior right mini-thoracotomy was also performed for achievement of liver debridement. Following extensive discussion regarding risk of mortality due to underlying disease progression, extensive comorbid infection, and inability to attain effective immune reconstitution in the face of heavy leukemic burden in the marrow, the decision was made to proceed with tisagenlecleucel. Lymphodepleting chemotherapy (fludarabine (dose: 30 mg/m2/day) and cytarabine (dose: 1 g/m2/day), modified due to severe lymphocytopenia and known alkylator-resistant disease) was administered over 2 days. At the time of infusion, the patient was normotensive, though still febrile and hypoxic (on supplemental nasal cannula) with three chest tubes in place following thoracotomy. The patient tolerated tisagenlecleucel infusion. Figure 1 Contrast-enhanced CT scans for both patients before tisagenlecleucel and after tisagenlecleucel infusion. (A) Patient 1: CT scans performed at diagnosis and following treatment. Axial (top left) and sagittal (bottom left) images at diagnosis demonstrate a lenticular hypodense structure (yellow arrows) centered at, and Inseparable from, the abnormally thickened right diaphragm (dashed arrows). This causes invasion or indentation of the adjacent posterior right hepatic contour. A small tubular air-filled component is present within the diaphragm (red arrows). Subtle asymmetric enlargement of the right M raises the question of chest wall involvement. There is consolidation of the adjacent right lung (). Post-treatment axial (top right) and sagittal (bottom right) images demonstrate resolution of the hypodense structure, with a residual thick-walled enhancing fluid collection along the diaphragm (yellow arrows). Note improved contour of the posterior right liver. Anastomotic sutures (red arrow) and a small left pleural effusion () are present. (B) Patient 2: CT scans performed at diagnosis and following treatment. Coronal (top left) and sagittal (bottom left) images prior to treatment demonstrate mildly enhancing soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A small amount of free fluid (*) is present. Post-treatment coronal (top right) and sagittal (bottom right) images demonstrate enlargement of the soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A, aorta; I, intrahepatic inferior vena cava; M, paraspinal muscle. Fever frequency and intensity increased several days after infusion, consistent with CRS. The patient remained normotensive with minimal oxygen requirement. On postinfusion day 8, tocilizumab was administered for a temperature of 42.9°C. Fever returned 12 hours later but eventually resolved 7 days after tocilizumab administration (figure 2A). No other signs or symptoms of CRS materialized. To help decide between palliative therapy and continued care with curative intent, an early response evaluation was performed 13 days after infusion. Bone marrow obtained at that time showed acellular morphology with 0.02% leukemia by flow cytometry. Given the low leukemic burden in the bone marrow and anticipated low likelihood of recurrent CRS, attention was turned to supporting hematopoiesis, which would be required to fully eradicate the invasive fungal infection. With this, granulocyte-macrophage colony-stimulating factor (GM-CSF) was initiated and granulocyte infusions were resumed. A repeat marrow was performed on day 28 postinfusion and showed a hypocellular marrow with no immunophenotypic evidence of disease. Due to persistent cytopenias and marrow hypocellularity, consideration was given to a CD34+ stem cell-selected boost from the patient’s mother (stem cell donor); by day+34 post-infusion, however, the patient achieved a spontaneous rise in his absolute neutrophil count. Granulocyte infusions were discontinued, and stem cell donation was canceled. The patient was discharged from the hospital 39 days after infusion. He remained on isavuconazonium, liposomal amphotericin B, and GM-CSF at time of discharge. Fifty days after infusion, GM-CSF was discontinued (figure 3A); no significant side effects were observed due to the use of GM-CSF. Figure 2 Temperature, treatment, ferritin levels, and CRP levels before and after tisagenlecleucel infusion. (A) Patient 1 temperature, treatment, ferritin levels, and CRP levels. (B) Patient 2 temperature, treatment, ferritin levels, and CRP levels. CRRT, continuous renal replacement therapy; CRP, C reactive protein; Cy, cyclophosphamide; ECMO, extracorporeal membrane oxygenation; Flu, fludarabine; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; LDC, lymphodepleting chemotherapy. Figure 3 Timeline of infections and treatment throughout the tisagenlecleucel therapy process. (A) Timeline for patient 1. aDay 8 of cefepime found muscle swelling and fungal elements in the liver; thus, the patient underwent surgery (right video-assisted thorascopic surgery with right lower lobe lung wedge resection, right thoracotomy, diaphragm biopsy, and right liver lobe wedge resection). bDiscontinued 2 days before infusion and restarted 14 days after until end of September 2018. cVideo-assisted thorascopic surgery in which a small portion of the right middle lung lobe was resected from the pleura and showed negative fungal elements. Lesion was not patched due to the potential of fungal growth. dOngoing observation. (B) Timeline for patient 2. aDelayed to December 31 due to infection. bPositive for rhinovirus/enterovirus, adenovirus, and BK virus, but remained asymptomatic. cContinued throughout blast count recovery. CRRT, continuous renal replacement therapy; GM-CSF, granulocyte-macrophage colony-stimulating factor; IVIG, intravenous immunoglobulin; LD, lymphodepleting; VA-ECMO, venoarterial extracorporeal membrane oxygenation. Forty-nine days after infusion, an additional thoracoscopic surgery was performed to resect residual necrotic lung; no fungal elements were detected at that time (figure 1A), and antifungal therapy was narrowed to monotherapy with isavuconazonium. The patient returned to his country of origin 76 days after infusion and remains in remission 19 months after infusion. Patient 2 A preschool-age Hispanic female was diagnosed with pre-B-ALL 14 months before tisagenlecleucel infusion and was treated according to the National Mexican Protocol, initially with achievement of complete remission (see online supplemental file). She developed relapsed disease 7 months prior to tisagenlecleucel infusion. She underwent reinduction chemotherapy, blinatumomab, and focal radiation; despite these therapies, bone marrow evaluation continued to show active disease 1 year after diagnosis, at which time she was referred for tisagenlecleucel infusion. The patient underwent leukapheresis 1 month prior to tisagenlecleucel infusion. She received bridging chemotherapy of 6-mercaptopurine and methotrexate and underwent lymphodepleting chemotherapy of fludarabine (30 mg/m2/dose for 3 days) and cyclophosphamide (500 mg/m2/dose for 2 days) 18 days prior to tisagenlecleucel infusion. Within 3 days of starting lymphodepleting chemotherapy, while neutropenic, she developed severe septic shock (extended spectrum beta-lactamase-positive and ertapenem-resistant Escherichia coli) with need for broad-spectrum antibiotics, steroids, inotropes, ventilatory support, 4 days of continuous renal replacement therapy and 5 days of venoarterial extracorporeal membrane oxygenation (VA-ECMO). VA-ECMO was discontinued 11 days prior to tisagenlecleucel infusion. Shortly thereafter, the patient developed recurrent fever. A CT scan obtained for evaluation of an infectious nidus showed pulmonary opacities with tree-in-bud appearance (possible fungal etiology) and a small intra-abdominal abscess/phlegmon. Given these findings, she was started on posaconazole in addition to existing broad-spectrum antimicrobials (figure 2B). With consideration given to disease burden (11% circulating leukemic blasts on day prior to infusion), optimal antifungal/antimicrobial therapy, and stable clinical exam, the decision was made to proceed with tisagenlecleucel infusion (figure 1B). At the time of infusion, respiratory failure was resolved (without need for non-invasive ventilatory support) and the patient was hemodynamically stable. She remained on amlodipine for management of underlying hypertension, but all inotropic support was discontinued. An echocardiogram (obtained 4 days preinfusion) showed qualitatively normal right ventricular systolic function and mild global hypokinesis of the left ventricle (ejection fraction (EF) 49%, shortening fraction (SF) 32%). The patient’s postinfusion course was complicated by CRS (characterized by fever, cardiac dysfunction, fluid overload, and respiratory insufficiency) and extensive lymphadenopathy (figure 1B) with associated renovascular compromise. She received two doses of tocilizumab on day+21 given 12 hours apart with some clinical improvement, though ultimately with need for resumption of non-invasive ventilatory support and milrinone due to depressed left ventricular systolic function (EF 45%, SF 21.5%). Given the unclear nature of the patient’s aforementioned extensive adenopathy and inability to exclude disease progression, a bone marrow biopsy and aspirate were completed on day 21 postinfusion; it revealed persistent marrow disease, though notably with numerous CD3+ T lymphocytes surrounding visualized blast clusters (figure 4A–E). Follow-up biopsy and aspirate obtained 31 days postinfusion showed extensive fibrosis with no morphological or immunophenotypic evidence of disease (figure 4F). The patient was discharged from the hospital 53 days after tisagenlecleucel infusion. An echocardiogram obtained prior to discharge showed mildly depressed left ventricle systolic function (EF 49%, SF 26%) with mild left ventricular dilation. At the time of discharge (day+54 postinfusion) and on transfer home to her country of origin (day+58), she remained on amlodipine for management of hypertension but was otherwise on no cardiac remodeling medications. She remained on granulocyte colony-stimulating factor for management of severe neutropenia and was transfusion-dependent (packed red blood cells and platelets). In the setting of severe neutropenia, levofloxacin and voriconazole were also continued for infection prophylaxis. Figure 4 Bone marrow biopsy for patient 2 before and after tisagenlecleucel. (A–F) Pathology of biopsies for patient 2. Pathology of the bone marrow at 21 days (A–E) and 31 days (F) after infusion. (A, B) Blasts in the bone marrow exhibited karyorrhexis (blue arrows) mixed with intact peripheral cells 21 days after infusion. (C) CD3+ T cells surrounding and infiltrating leukemic blasts. Blue arrows indicate CD3-negative blast cells. (D) CD19 expression on the blasts. (E) Dual CD3-CD10+ immunostaining highlight T cells in brown (black arrows) and blasts in red (blue arrows). (F) Bone marrow at day 31, depleted of hematopoietic cells. The patient returned to her country of origin 58 days after infusion. Unfortunately, she expired 208 days after infusion due to cardiac arrest of unclear etiology (figure 3B). An autopsy was not obtained. Her last bone marrow evaluation was completed 3 weeks prior to death and showed no morphological or immunophenotypic evidence of disease. Discussion Here, we describe two patients with heavily pretreated r/r B-ALL who were safely infused with tisagenlecleucel despite elevated inflammatory markers, heavy leukemic burden, significant infection, and multiple comorbidities. While our center has experience with infusion of CAR-T cells dating back to 2012 and physician experience with infusions dating to 2005, tisagenlecleucel infusions are and will be the first such infusions for many centers. The complex management of patients prior to, during, and after infusion can be a source of significant concern for many providers, particularly in the presence of underlying infection, evolving disease burden, and/or other confounding variables that increase risk for severe CRS. Early recommendations to sites regarding conditions for infusion and CRS management on the pivotal multisite ELIANA trial of tisagenlecleucel were based on adult and pediatric experience at a single institution. During the trial, experienced providers were frequently consulted by those centers or providers with less experience in CAR-T cell infusion when challenged by cases for which the existing CRS management guidelines did not provide clear guidance on how to proceed (ie, a provider may inquire about tocilizumab for a patient with 5 days of persistent fever to 40°C without other signs or symptoms of CRS). Earlier in the ELIANA trial, there was significant concern that early CRS intervention might lead to diminished efficacy. Through increased patient experience in both trial and real-world settings, however, much has been learned about patient management, including management of mild to severe CRS and neurological toxicity.4 As the number of sites administering tisagenlecleucel expands and a wide range of real-world experience is obtained, it will be difficult to implement rapid changes to official guidelines. In the ELIANA trial, 43% of patients experienced infections within 8 weeks of tisagenlecleucel infusion, and 24% were severe infections (grade 3/4).2 In a few patients, prolonged severe neutropenia led to severe (grade 3 human herpesvirus 6 encephalitis) and fatal (encephalitis and systemic mycosis) infections. Analysis of real-world data in patients with B-ALL from the Center for International Blood and Marrow Transplant Research (CIBMTR) patient registry reported a similar incidence of postinfusion infections (42%), with one fatal bacterial infection.5 Furthermore, management of CRS with short-term tocilizumab therapy (IL-6 blockade) does not appear to increase the risk of infection6; however, it is important to consider that immune responses to invasive fungal infections could involve IL-67 8 and therefore may respond differently to tocilizumab therapy than was reported for our patient. Taken together, these clinical trial and real-world data highlight the known postinfusion infection risk in these patients; however, existing data fail to provide insight into how patients with preinfusion infections might be managed. Expanding the existing literature to further describe management of complex patients who, in some respects, challenge the current guidelines for infusion will be important for improving access to therapy for patients who lack other options for potential durable remission. Technically, the patients reported here were infused with tisagenlecleucel outside of label guidelines due to the presence of elevated inflammatory markers and active infection.1 For patient 1, CRS manifestations were limited to only fever without hypotension or progressive renal or pulmonary involvement. Response of B-ALL to tisagenlecleucel was not compromised by the treatment of CRS at this stage with tocilizumab. For patient 2, CRS was characterized by fever, third-spacing, cardiac dysfunction, and marked lymphadenopathy with secondary renal vascular compromise (suspected to be driven by CAR-T cell expansion). Cardiac dysfunction due to CRS has been observed in children undergoing CAR-T cell therapy, and risk of dysfunction may be increased in patients who have pre-existing cardiac abnormalities; persistent dysfunction remains uncommon, however.9 10 For patient 2, improvement in cardiac function was observed following resolution of CRS, though with unexpected death due to cardiac arrest greater than 200 days after infusion. The etiology of this arrest remains unknown at this time. Through experience gained treating CRS in patients who were part of the ELIANA study, we felt relatively comfortable that we could control CRS and therefore infused both patients under circumstances in which other providers might not have. Nevertheless, tisagenlecleucel infusion was only carried out after extensive counseling regarding the possibility of worst-case scenarios and with serious consideration also given to transport home for palliative measures. In both cases, significant discussions regarding potential life-threatening outcomes were had with patients, families, and medical providers prior to infusion. Conclusions Publication of these case studies and others may guide providers and families as they face similarly challenging circumstances in the future. In addition, they highlight the importance of clear communication with patients, families, and medical team members about potentially lethal adverse events of CAR-T cell therapy when infused in the setting of concurrent life-threatening comorbidities. While these cases represent successful infection management during CAR-T cell therapy, they should not be viewed as typical or even likely outcomes. Treatment of both patients was carried out in a setting with extensive institutional knowledge by an experienced team with the ability to customize standard protocols. Additional reporting of outcomes in patients with active infections via the CIBMTR patient registry is needed to confirm the generalizability of our approach and to better delineate benefit–risk assessment in these patients. Careful reporting on patient experiences and outcomes in the postcommercialization era of tisagenlecleucel will help to ensure safe access to potentially curative therapy for more patients. Ethics statements Patient consent for publication Obtained. Ethics approval This is a retrospective case series for which institutional review board permissions were granted and results were pulled from patients’ charts. Correction notice: This article has been corrected since it first published. The provenance and peer review statement has been included. Contributors: All authors treated patients and contributed to data collection and interpretation. EMH and GDM provided data and helped write the first draft. All authors were involved in revising the manuscript, and read and approved the final version. Funding: Medical writing support was provided by Briana Dye, PhD, of Healthcare Consultancy Group, and was funded by Novartis Pharmaceuticals Corporation. Competing interests: DEY has been an investigator on studies supported by Astellas, Chimerix, Merck, Pfizer, and Viracor-Eurofins. KJA is on the Speaker’s Bureau for and receives support from Novartis Pharmaceuticals. GDM is a consultant and is on the Speaker’s Bureau for Novartis Pharmaceuticals. EMH, RKG, AAA, GSM, SDS, TGF, IAA, WL, and RJH declare that they have no competing interests. Provenance and peer review: Not commissioned; externally peer reviewed. Supplemental material: This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.	8 DOSES	DrugDosageText	CC BY-NC	33472856	19,003,489	2021-01
What was the dosage of drug 'BLINATUMOMAB'?	Tisagenlecleucel infusion in patients with relapsed/refractory ALL and concurrent serious infection. Tisagenlecleucel, an anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy, has demonstrated durable efficacy and a manageable safety profile in pediatric and young adult patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) in the ELIANA pivotal trial and real-world experience. Experience from investigator-led studies prior to ELIANA suggests that infections and inflammatory conditions may exacerbate the severity of cytokine release syndrome (CRS) associated with CAR-T cell therapy, leading to extreme caution and strong restrictions for on-study and commercial infusion of tisagenlecleucel in patients with active infection. CRS intervention with interleukin (IL)-6 blockade and/or steroid therapy was introduced late in the course during clinical trials due to concern for potential negative effect on efficacy and persistence. However, earlier CRS intervention is now viewed more favorably. Earlier intervention and consistency in management between providers may promote broader use of tisagenlecleucel, including potential curative therapy in patients who require remission and recovery of hematopoiesis for management of severe infection. Patient 1 was diagnosed with B-ALL at 23 years old. Fourteen days before tisagenlecleucel infusion, the patient developed fever and neutropenia and was diagnosed with invasive Mucorales infection and BK virus hemorrhagic cystitis. Aggressive measures were instituted to control infection and to manage prolonged cytopenias during CAR-T cell manufacturing. Adverse events, including CRS, were manageable despite elevated inflammatory markers and active infection. The patient attained remission and recovered hematopoiesis, and infections resolved. The patient remains in remission ≥1 year postinfusion.Patient 2 was diagnosed with pre-B-ALL at preschool age. She developed severe septic shock 3 days postinitiation of lymphodepleting chemotherapy. After receiving tisagenlecleucel, she experienced CRS with cardiac dysfunction and extensive lymphadenopathy leading to renovascular compromise. The patient attained remission and was discharged in good condition to her country of origin. She remained in remission but expired on day 208 postinfusion due to cardiac arrest of unclear etiology. Infusion was feasible, and toxicity related to tisagenlecleucel was manageable despite active infections and concurrent inflammation, allowing attainment of remission in otherwise refractory pediatric/young adult ALL. This may lead to consideration of tisagenlecleucel as a potential curative therapy in patients with managed active infections. pmcBackground Anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy (tisagenlecleucel) has demonstrated durable efficacy and manageable safety for pediatric and young adult patients with relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL).1–3 In the single-arm, phase 2 ELIANA study (NCT02435849), 79 patients with r/r B-ALL (age range, 3–24 years) were infused with a single dose of tisagenlecleucel. The overall remission rate (complete remission and complete remission with incomplete blood count recovery) was 82% within 3 months. Adverse events of special interest included cytokine release syndrome (CRS, 77%) and neurological toxicities (39%).3 Patients with active infections were not enrolled and infusion was held for cases of uncontrolled infection or inflammation. Current US prescribing information warns against administration of tisagenlecleucel to patients with active infection or inflammatory conditions.1 However, there remains a lack of data for CAR-T cell therapy in patients with active, managed infections. Furthermore, there are varying levels of comfort in treating patients with different levels of disease burden, infection types, and comorbidities. The publication of clinician and patient experiences with difficult cases should benefit providers treating patients in need of potentially curative therapy but who may be delayed or denied therapy based on prior guidelines for infusion. Herein, we describe two patients with r/r B-ALL and concurrent infection and/or organ dysfunction who were safely infused with and responded to tisagenlecleucel. Case presentation Patient 1 A 23-year-old male was diagnosed with B-ALL 18 months before tisagenlecleucel infusion. His disease relapsed after standard and salvage chemotherapy with development of refractory disease following haploidentical hematopoietic stem cell transplant. An additional text file includes further treatment history (see online supplemental file). The patient was evaluated for tisagenlecleucel and leukapheresis occurred 17 months after diagnosis. Bone marrow evaluation after bridging chemotherapy revealed 95% blasts in a normocellular marrow. 10.1136/jitc-2020-001225.supp1 Supplementary data Fourteen days preinfusion, the patient was admitted for fever/neutropenia. Due to persistent fevers, a CT scan was obtained, which demonstrated findings concerning for fungal infection. He underwent right lung thoracotomy and diaphragm biopsy and was diagnosed with invasive Mucorales infection with involvement of the right lower lung lobe, diaphragm, and liver (figure 1A). Therapy was initiated with intravenous liposomal amphotericin B, micafungin, and posaconazole (switched to isavuconazonium sulfate after 4 days); intrathoracic amphotericin B deoxycholate and granulocyte infusions were also administered. The patient remained febrile with severe pancytopenia and severe concurrent BK virus hemorrhagic cystitis. He underwent thorascopic resection of the necrotic lung by wedge resection. A posterior right mini-thoracotomy was also performed for achievement of liver debridement. Following extensive discussion regarding risk of mortality due to underlying disease progression, extensive comorbid infection, and inability to attain effective immune reconstitution in the face of heavy leukemic burden in the marrow, the decision was made to proceed with tisagenlecleucel. Lymphodepleting chemotherapy (fludarabine (dose: 30 mg/m2/day) and cytarabine (dose: 1 g/m2/day), modified due to severe lymphocytopenia and known alkylator-resistant disease) was administered over 2 days. At the time of infusion, the patient was normotensive, though still febrile and hypoxic (on supplemental nasal cannula) with three chest tubes in place following thoracotomy. The patient tolerated tisagenlecleucel infusion. Figure 1 Contrast-enhanced CT scans for both patients before tisagenlecleucel and after tisagenlecleucel infusion. (A) Patient 1: CT scans performed at diagnosis and following treatment. Axial (top left) and sagittal (bottom left) images at diagnosis demonstrate a lenticular hypodense structure (yellow arrows) centered at, and Inseparable from, the abnormally thickened right diaphragm (dashed arrows). This causes invasion or indentation of the adjacent posterior right hepatic contour. A small tubular air-filled component is present within the diaphragm (red arrows). Subtle asymmetric enlargement of the right M raises the question of chest wall involvement. There is consolidation of the adjacent right lung (). Post-treatment axial (top right) and sagittal (bottom right) images demonstrate resolution of the hypodense structure, with a residual thick-walled enhancing fluid collection along the diaphragm (yellow arrows). Note improved contour of the posterior right liver. Anastomotic sutures (red arrow) and a small left pleural effusion () are present. (B) Patient 2: CT scans performed at diagnosis and following treatment. Coronal (top left) and sagittal (bottom left) images prior to treatment demonstrate mildly enhancing soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A small amount of free fluid (*) is present. Post-treatment coronal (top right) and sagittal (bottom right) images demonstrate enlargement of the soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A, aorta; I, intrahepatic inferior vena cava; M, paraspinal muscle. Fever frequency and intensity increased several days after infusion, consistent with CRS. The patient remained normotensive with minimal oxygen requirement. On postinfusion day 8, tocilizumab was administered for a temperature of 42.9°C. Fever returned 12 hours later but eventually resolved 7 days after tocilizumab administration (figure 2A). No other signs or symptoms of CRS materialized. To help decide between palliative therapy and continued care with curative intent, an early response evaluation was performed 13 days after infusion. Bone marrow obtained at that time showed acellular morphology with 0.02% leukemia by flow cytometry. Given the low leukemic burden in the bone marrow and anticipated low likelihood of recurrent CRS, attention was turned to supporting hematopoiesis, which would be required to fully eradicate the invasive fungal infection. With this, granulocyte-macrophage colony-stimulating factor (GM-CSF) was initiated and granulocyte infusions were resumed. A repeat marrow was performed on day 28 postinfusion and showed a hypocellular marrow with no immunophenotypic evidence of disease. Due to persistent cytopenias and marrow hypocellularity, consideration was given to a CD34+ stem cell-selected boost from the patient’s mother (stem cell donor); by day+34 post-infusion, however, the patient achieved a spontaneous rise in his absolute neutrophil count. Granulocyte infusions were discontinued, and stem cell donation was canceled. The patient was discharged from the hospital 39 days after infusion. He remained on isavuconazonium, liposomal amphotericin B, and GM-CSF at time of discharge. Fifty days after infusion, GM-CSF was discontinued (figure 3A); no significant side effects were observed due to the use of GM-CSF. Figure 2 Temperature, treatment, ferritin levels, and CRP levels before and after tisagenlecleucel infusion. (A) Patient 1 temperature, treatment, ferritin levels, and CRP levels. (B) Patient 2 temperature, treatment, ferritin levels, and CRP levels. CRRT, continuous renal replacement therapy; CRP, C reactive protein; Cy, cyclophosphamide; ECMO, extracorporeal membrane oxygenation; Flu, fludarabine; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; LDC, lymphodepleting chemotherapy. Figure 3 Timeline of infections and treatment throughout the tisagenlecleucel therapy process. (A) Timeline for patient 1. aDay 8 of cefepime found muscle swelling and fungal elements in the liver; thus, the patient underwent surgery (right video-assisted thorascopic surgery with right lower lobe lung wedge resection, right thoracotomy, diaphragm biopsy, and right liver lobe wedge resection). bDiscontinued 2 days before infusion and restarted 14 days after until end of September 2018. cVideo-assisted thorascopic surgery in which a small portion of the right middle lung lobe was resected from the pleura and showed negative fungal elements. Lesion was not patched due to the potential of fungal growth. dOngoing observation. (B) Timeline for patient 2. aDelayed to December 31 due to infection. bPositive for rhinovirus/enterovirus, adenovirus, and BK virus, but remained asymptomatic. cContinued throughout blast count recovery. CRRT, continuous renal replacement therapy; GM-CSF, granulocyte-macrophage colony-stimulating factor; IVIG, intravenous immunoglobulin; LD, lymphodepleting; VA-ECMO, venoarterial extracorporeal membrane oxygenation. Forty-nine days after infusion, an additional thoracoscopic surgery was performed to resect residual necrotic lung; no fungal elements were detected at that time (figure 1A), and antifungal therapy was narrowed to monotherapy with isavuconazonium. The patient returned to his country of origin 76 days after infusion and remains in remission 19 months after infusion. Patient 2 A preschool-age Hispanic female was diagnosed with pre-B-ALL 14 months before tisagenlecleucel infusion and was treated according to the National Mexican Protocol, initially with achievement of complete remission (see online supplemental file). She developed relapsed disease 7 months prior to tisagenlecleucel infusion. She underwent reinduction chemotherapy, blinatumomab, and focal radiation; despite these therapies, bone marrow evaluation continued to show active disease 1 year after diagnosis, at which time she was referred for tisagenlecleucel infusion. The patient underwent leukapheresis 1 month prior to tisagenlecleucel infusion. She received bridging chemotherapy of 6-mercaptopurine and methotrexate and underwent lymphodepleting chemotherapy of fludarabine (30 mg/m2/dose for 3 days) and cyclophosphamide (500 mg/m2/dose for 2 days) 18 days prior to tisagenlecleucel infusion. Within 3 days of starting lymphodepleting chemotherapy, while neutropenic, she developed severe septic shock (extended spectrum beta-lactamase-positive and ertapenem-resistant Escherichia coli) with need for broad-spectrum antibiotics, steroids, inotropes, ventilatory support, 4 days of continuous renal replacement therapy and 5 days of venoarterial extracorporeal membrane oxygenation (VA-ECMO). VA-ECMO was discontinued 11 days prior to tisagenlecleucel infusion. Shortly thereafter, the patient developed recurrent fever. A CT scan obtained for evaluation of an infectious nidus showed pulmonary opacities with tree-in-bud appearance (possible fungal etiology) and a small intra-abdominal abscess/phlegmon. Given these findings, she was started on posaconazole in addition to existing broad-spectrum antimicrobials (figure 2B). With consideration given to disease burden (11% circulating leukemic blasts on day prior to infusion), optimal antifungal/antimicrobial therapy, and stable clinical exam, the decision was made to proceed with tisagenlecleucel infusion (figure 1B). At the time of infusion, respiratory failure was resolved (without need for non-invasive ventilatory support) and the patient was hemodynamically stable. She remained on amlodipine for management of underlying hypertension, but all inotropic support was discontinued. An echocardiogram (obtained 4 days preinfusion) showed qualitatively normal right ventricular systolic function and mild global hypokinesis of the left ventricle (ejection fraction (EF) 49%, shortening fraction (SF) 32%). The patient’s postinfusion course was complicated by CRS (characterized by fever, cardiac dysfunction, fluid overload, and respiratory insufficiency) and extensive lymphadenopathy (figure 1B) with associated renovascular compromise. She received two doses of tocilizumab on day+21 given 12 hours apart with some clinical improvement, though ultimately with need for resumption of non-invasive ventilatory support and milrinone due to depressed left ventricular systolic function (EF 45%, SF 21.5%). Given the unclear nature of the patient’s aforementioned extensive adenopathy and inability to exclude disease progression, a bone marrow biopsy and aspirate were completed on day 21 postinfusion; it revealed persistent marrow disease, though notably with numerous CD3+ T lymphocytes surrounding visualized blast clusters (figure 4A–E). Follow-up biopsy and aspirate obtained 31 days postinfusion showed extensive fibrosis with no morphological or immunophenotypic evidence of disease (figure 4F). The patient was discharged from the hospital 53 days after tisagenlecleucel infusion. An echocardiogram obtained prior to discharge showed mildly depressed left ventricle systolic function (EF 49%, SF 26%) with mild left ventricular dilation. At the time of discharge (day+54 postinfusion) and on transfer home to her country of origin (day+58), she remained on amlodipine for management of hypertension but was otherwise on no cardiac remodeling medications. She remained on granulocyte colony-stimulating factor for management of severe neutropenia and was transfusion-dependent (packed red blood cells and platelets). In the setting of severe neutropenia, levofloxacin and voriconazole were also continued for infection prophylaxis. Figure 4 Bone marrow biopsy for patient 2 before and after tisagenlecleucel. (A–F) Pathology of biopsies for patient 2. Pathology of the bone marrow at 21 days (A–E) and 31 days (F) after infusion. (A, B) Blasts in the bone marrow exhibited karyorrhexis (blue arrows) mixed with intact peripheral cells 21 days after infusion. (C) CD3+ T cells surrounding and infiltrating leukemic blasts. Blue arrows indicate CD3-negative blast cells. (D) CD19 expression on the blasts. (E) Dual CD3-CD10+ immunostaining highlight T cells in brown (black arrows) and blasts in red (blue arrows). (F) Bone marrow at day 31, depleted of hematopoietic cells. The patient returned to her country of origin 58 days after infusion. Unfortunately, she expired 208 days after infusion due to cardiac arrest of unclear etiology (figure 3B). An autopsy was not obtained. Her last bone marrow evaluation was completed 3 weeks prior to death and showed no morphological or immunophenotypic evidence of disease. Discussion Here, we describe two patients with heavily pretreated r/r B-ALL who were safely infused with tisagenlecleucel despite elevated inflammatory markers, heavy leukemic burden, significant infection, and multiple comorbidities. While our center has experience with infusion of CAR-T cells dating back to 2012 and physician experience with infusions dating to 2005, tisagenlecleucel infusions are and will be the first such infusions for many centers. The complex management of patients prior to, during, and after infusion can be a source of significant concern for many providers, particularly in the presence of underlying infection, evolving disease burden, and/or other confounding variables that increase risk for severe CRS. Early recommendations to sites regarding conditions for infusion and CRS management on the pivotal multisite ELIANA trial of tisagenlecleucel were based on adult and pediatric experience at a single institution. During the trial, experienced providers were frequently consulted by those centers or providers with less experience in CAR-T cell infusion when challenged by cases for which the existing CRS management guidelines did not provide clear guidance on how to proceed (ie, a provider may inquire about tocilizumab for a patient with 5 days of persistent fever to 40°C without other signs or symptoms of CRS). Earlier in the ELIANA trial, there was significant concern that early CRS intervention might lead to diminished efficacy. Through increased patient experience in both trial and real-world settings, however, much has been learned about patient management, including management of mild to severe CRS and neurological toxicity.4 As the number of sites administering tisagenlecleucel expands and a wide range of real-world experience is obtained, it will be difficult to implement rapid changes to official guidelines. In the ELIANA trial, 43% of patients experienced infections within 8 weeks of tisagenlecleucel infusion, and 24% were severe infections (grade 3/4).2 In a few patients, prolonged severe neutropenia led to severe (grade 3 human herpesvirus 6 encephalitis) and fatal (encephalitis and systemic mycosis) infections. Analysis of real-world data in patients with B-ALL from the Center for International Blood and Marrow Transplant Research (CIBMTR) patient registry reported a similar incidence of postinfusion infections (42%), with one fatal bacterial infection.5 Furthermore, management of CRS with short-term tocilizumab therapy (IL-6 blockade) does not appear to increase the risk of infection6; however, it is important to consider that immune responses to invasive fungal infections could involve IL-67 8 and therefore may respond differently to tocilizumab therapy than was reported for our patient. Taken together, these clinical trial and real-world data highlight the known postinfusion infection risk in these patients; however, existing data fail to provide insight into how patients with preinfusion infections might be managed. Expanding the existing literature to further describe management of complex patients who, in some respects, challenge the current guidelines for infusion will be important for improving access to therapy for patients who lack other options for potential durable remission. Technically, the patients reported here were infused with tisagenlecleucel outside of label guidelines due to the presence of elevated inflammatory markers and active infection.1 For patient 1, CRS manifestations were limited to only fever without hypotension or progressive renal or pulmonary involvement. Response of B-ALL to tisagenlecleucel was not compromised by the treatment of CRS at this stage with tocilizumab. For patient 2, CRS was characterized by fever, third-spacing, cardiac dysfunction, and marked lymphadenopathy with secondary renal vascular compromise (suspected to be driven by CAR-T cell expansion). Cardiac dysfunction due to CRS has been observed in children undergoing CAR-T cell therapy, and risk of dysfunction may be increased in patients who have pre-existing cardiac abnormalities; persistent dysfunction remains uncommon, however.9 10 For patient 2, improvement in cardiac function was observed following resolution of CRS, though with unexpected death due to cardiac arrest greater than 200 days after infusion. The etiology of this arrest remains unknown at this time. Through experience gained treating CRS in patients who were part of the ELIANA study, we felt relatively comfortable that we could control CRS and therefore infused both patients under circumstances in which other providers might not have. Nevertheless, tisagenlecleucel infusion was only carried out after extensive counseling regarding the possibility of worst-case scenarios and with serious consideration also given to transport home for palliative measures. In both cases, significant discussions regarding potential life-threatening outcomes were had with patients, families, and medical providers prior to infusion. Conclusions Publication of these case studies and others may guide providers and families as they face similarly challenging circumstances in the future. In addition, they highlight the importance of clear communication with patients, families, and medical team members about potentially lethal adverse events of CAR-T cell therapy when infused in the setting of concurrent life-threatening comorbidities. While these cases represent successful infection management during CAR-T cell therapy, they should not be viewed as typical or even likely outcomes. Treatment of both patients was carried out in a setting with extensive institutional knowledge by an experienced team with the ability to customize standard protocols. Additional reporting of outcomes in patients with active infections via the CIBMTR patient registry is needed to confirm the generalizability of our approach and to better delineate benefit–risk assessment in these patients. Careful reporting on patient experiences and outcomes in the postcommercialization era of tisagenlecleucel will help to ensure safe access to potentially curative therapy for more patients. Ethics statements Patient consent for publication Obtained. Ethics approval This is a retrospective case series for which institutional review board permissions were granted and results were pulled from patients’ charts. Correction notice: This article has been corrected since it first published. The provenance and peer review statement has been included. Contributors: All authors treated patients and contributed to data collection and interpretation. EMH and GDM provided data and helped write the first draft. All authors were involved in revising the manuscript, and read and approved the final version. Funding: Medical writing support was provided by Briana Dye, PhD, of Healthcare Consultancy Group, and was funded by Novartis Pharmaceuticals Corporation. Competing interests: DEY has been an investigator on studies supported by Astellas, Chimerix, Merck, Pfizer, and Viracor-Eurofins. KJA is on the Speaker’s Bureau for and receives support from Novartis Pharmaceuticals. GDM is a consultant and is on the Speaker’s Bureau for Novartis Pharmaceuticals. EMH, RKG, AAA, GSM, SDS, TGF, IAA, WL, and RJH declare that they have no competing interests. Provenance and peer review: Not commissioned; externally peer reviewed. Supplemental material: This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.	2 DOSES	DrugDosageText	CC BY-NC	33472856	19,003,489	2021-01
What was the dosage of drug 'DEXAMETHASONE'?	Tisagenlecleucel infusion in patients with relapsed/refractory ALL and concurrent serious infection. Tisagenlecleucel, an anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy, has demonstrated durable efficacy and a manageable safety profile in pediatric and young adult patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) in the ELIANA pivotal trial and real-world experience. Experience from investigator-led studies prior to ELIANA suggests that infections and inflammatory conditions may exacerbate the severity of cytokine release syndrome (CRS) associated with CAR-T cell therapy, leading to extreme caution and strong restrictions for on-study and commercial infusion of tisagenlecleucel in patients with active infection. CRS intervention with interleukin (IL)-6 blockade and/or steroid therapy was introduced late in the course during clinical trials due to concern for potential negative effect on efficacy and persistence. However, earlier CRS intervention is now viewed more favorably. Earlier intervention and consistency in management between providers may promote broader use of tisagenlecleucel, including potential curative therapy in patients who require remission and recovery of hematopoiesis for management of severe infection. Patient 1 was diagnosed with B-ALL at 23 years old. Fourteen days before tisagenlecleucel infusion, the patient developed fever and neutropenia and was diagnosed with invasive Mucorales infection and BK virus hemorrhagic cystitis. Aggressive measures were instituted to control infection and to manage prolonged cytopenias during CAR-T cell manufacturing. Adverse events, including CRS, were manageable despite elevated inflammatory markers and active infection. The patient attained remission and recovered hematopoiesis, and infections resolved. The patient remains in remission ≥1 year postinfusion.Patient 2 was diagnosed with pre-B-ALL at preschool age. She developed severe septic shock 3 days postinitiation of lymphodepleting chemotherapy. After receiving tisagenlecleucel, she experienced CRS with cardiac dysfunction and extensive lymphadenopathy leading to renovascular compromise. The patient attained remission and was discharged in good condition to her country of origin. She remained in remission but expired on day 208 postinfusion due to cardiac arrest of unclear etiology. Infusion was feasible, and toxicity related to tisagenlecleucel was manageable despite active infections and concurrent inflammation, allowing attainment of remission in otherwise refractory pediatric/young adult ALL. This may lead to consideration of tisagenlecleucel as a potential curative therapy in patients with managed active infections. pmcBackground Anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy (tisagenlecleucel) has demonstrated durable efficacy and manageable safety for pediatric and young adult patients with relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL).1–3 In the single-arm, phase 2 ELIANA study (NCT02435849), 79 patients with r/r B-ALL (age range, 3–24 years) were infused with a single dose of tisagenlecleucel. The overall remission rate (complete remission and complete remission with incomplete blood count recovery) was 82% within 3 months. Adverse events of special interest included cytokine release syndrome (CRS, 77%) and neurological toxicities (39%).3 Patients with active infections were not enrolled and infusion was held for cases of uncontrolled infection or inflammation. Current US prescribing information warns against administration of tisagenlecleucel to patients with active infection or inflammatory conditions.1 However, there remains a lack of data for CAR-T cell therapy in patients with active, managed infections. Furthermore, there are varying levels of comfort in treating patients with different levels of disease burden, infection types, and comorbidities. The publication of clinician and patient experiences with difficult cases should benefit providers treating patients in need of potentially curative therapy but who may be delayed or denied therapy based on prior guidelines for infusion. Herein, we describe two patients with r/r B-ALL and concurrent infection and/or organ dysfunction who were safely infused with and responded to tisagenlecleucel. Case presentation Patient 1 A 23-year-old male was diagnosed with B-ALL 18 months before tisagenlecleucel infusion. His disease relapsed after standard and salvage chemotherapy with development of refractory disease following haploidentical hematopoietic stem cell transplant. An additional text file includes further treatment history (see online supplemental file). The patient was evaluated for tisagenlecleucel and leukapheresis occurred 17 months after diagnosis. Bone marrow evaluation after bridging chemotherapy revealed 95% blasts in a normocellular marrow. 10.1136/jitc-2020-001225.supp1 Supplementary data Fourteen days preinfusion, the patient was admitted for fever/neutropenia. Due to persistent fevers, a CT scan was obtained, which demonstrated findings concerning for fungal infection. He underwent right lung thoracotomy and diaphragm biopsy and was diagnosed with invasive Mucorales infection with involvement of the right lower lung lobe, diaphragm, and liver (figure 1A). Therapy was initiated with intravenous liposomal amphotericin B, micafungin, and posaconazole (switched to isavuconazonium sulfate after 4 days); intrathoracic amphotericin B deoxycholate and granulocyte infusions were also administered. The patient remained febrile with severe pancytopenia and severe concurrent BK virus hemorrhagic cystitis. He underwent thorascopic resection of the necrotic lung by wedge resection. A posterior right mini-thoracotomy was also performed for achievement of liver debridement. Following extensive discussion regarding risk of mortality due to underlying disease progression, extensive comorbid infection, and inability to attain effective immune reconstitution in the face of heavy leukemic burden in the marrow, the decision was made to proceed with tisagenlecleucel. Lymphodepleting chemotherapy (fludarabine (dose: 30 mg/m2/day) and cytarabine (dose: 1 g/m2/day), modified due to severe lymphocytopenia and known alkylator-resistant disease) was administered over 2 days. At the time of infusion, the patient was normotensive, though still febrile and hypoxic (on supplemental nasal cannula) with three chest tubes in place following thoracotomy. The patient tolerated tisagenlecleucel infusion. Figure 1 Contrast-enhanced CT scans for both patients before tisagenlecleucel and after tisagenlecleucel infusion. (A) Patient 1: CT scans performed at diagnosis and following treatment. Axial (top left) and sagittal (bottom left) images at diagnosis demonstrate a lenticular hypodense structure (yellow arrows) centered at, and Inseparable from, the abnormally thickened right diaphragm (dashed arrows). This causes invasion or indentation of the adjacent posterior right hepatic contour. A small tubular air-filled component is present within the diaphragm (red arrows). Subtle asymmetric enlargement of the right M raises the question of chest wall involvement. There is consolidation of the adjacent right lung (). Post-treatment axial (top right) and sagittal (bottom right) images demonstrate resolution of the hypodense structure, with a residual thick-walled enhancing fluid collection along the diaphragm (yellow arrows). Note improved contour of the posterior right liver. Anastomotic sutures (red arrow) and a small left pleural effusion () are present. (B) Patient 2: CT scans performed at diagnosis and following treatment. Coronal (top left) and sagittal (bottom left) images prior to treatment demonstrate mildly enhancing soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A small amount of free fluid (*) is present. Post-treatment coronal (top right) and sagittal (bottom right) images demonstrate enlargement of the soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A, aorta; I, intrahepatic inferior vena cava; M, paraspinal muscle. Fever frequency and intensity increased several days after infusion, consistent with CRS. The patient remained normotensive with minimal oxygen requirement. On postinfusion day 8, tocilizumab was administered for a temperature of 42.9°C. Fever returned 12 hours later but eventually resolved 7 days after tocilizumab administration (figure 2A). No other signs or symptoms of CRS materialized. To help decide between palliative therapy and continued care with curative intent, an early response evaluation was performed 13 days after infusion. Bone marrow obtained at that time showed acellular morphology with 0.02% leukemia by flow cytometry. Given the low leukemic burden in the bone marrow and anticipated low likelihood of recurrent CRS, attention was turned to supporting hematopoiesis, which would be required to fully eradicate the invasive fungal infection. With this, granulocyte-macrophage colony-stimulating factor (GM-CSF) was initiated and granulocyte infusions were resumed. A repeat marrow was performed on day 28 postinfusion and showed a hypocellular marrow with no immunophenotypic evidence of disease. Due to persistent cytopenias and marrow hypocellularity, consideration was given to a CD34+ stem cell-selected boost from the patient’s mother (stem cell donor); by day+34 post-infusion, however, the patient achieved a spontaneous rise in his absolute neutrophil count. Granulocyte infusions were discontinued, and stem cell donation was canceled. The patient was discharged from the hospital 39 days after infusion. He remained on isavuconazonium, liposomal amphotericin B, and GM-CSF at time of discharge. Fifty days after infusion, GM-CSF was discontinued (figure 3A); no significant side effects were observed due to the use of GM-CSF. Figure 2 Temperature, treatment, ferritin levels, and CRP levels before and after tisagenlecleucel infusion. (A) Patient 1 temperature, treatment, ferritin levels, and CRP levels. (B) Patient 2 temperature, treatment, ferritin levels, and CRP levels. CRRT, continuous renal replacement therapy; CRP, C reactive protein; Cy, cyclophosphamide; ECMO, extracorporeal membrane oxygenation; Flu, fludarabine; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; LDC, lymphodepleting chemotherapy. Figure 3 Timeline of infections and treatment throughout the tisagenlecleucel therapy process. (A) Timeline for patient 1. aDay 8 of cefepime found muscle swelling and fungal elements in the liver; thus, the patient underwent surgery (right video-assisted thorascopic surgery with right lower lobe lung wedge resection, right thoracotomy, diaphragm biopsy, and right liver lobe wedge resection). bDiscontinued 2 days before infusion and restarted 14 days after until end of September 2018. cVideo-assisted thorascopic surgery in which a small portion of the right middle lung lobe was resected from the pleura and showed negative fungal elements. Lesion was not patched due to the potential of fungal growth. dOngoing observation. (B) Timeline for patient 2. aDelayed to December 31 due to infection. bPositive for rhinovirus/enterovirus, adenovirus, and BK virus, but remained asymptomatic. cContinued throughout blast count recovery. CRRT, continuous renal replacement therapy; GM-CSF, granulocyte-macrophage colony-stimulating factor; IVIG, intravenous immunoglobulin; LD, lymphodepleting; VA-ECMO, venoarterial extracorporeal membrane oxygenation. Forty-nine days after infusion, an additional thoracoscopic surgery was performed to resect residual necrotic lung; no fungal elements were detected at that time (figure 1A), and antifungal therapy was narrowed to monotherapy with isavuconazonium. The patient returned to his country of origin 76 days after infusion and remains in remission 19 months after infusion. Patient 2 A preschool-age Hispanic female was diagnosed with pre-B-ALL 14 months before tisagenlecleucel infusion and was treated according to the National Mexican Protocol, initially with achievement of complete remission (see online supplemental file). She developed relapsed disease 7 months prior to tisagenlecleucel infusion. She underwent reinduction chemotherapy, blinatumomab, and focal radiation; despite these therapies, bone marrow evaluation continued to show active disease 1 year after diagnosis, at which time she was referred for tisagenlecleucel infusion. The patient underwent leukapheresis 1 month prior to tisagenlecleucel infusion. She received bridging chemotherapy of 6-mercaptopurine and methotrexate and underwent lymphodepleting chemotherapy of fludarabine (30 mg/m2/dose for 3 days) and cyclophosphamide (500 mg/m2/dose for 2 days) 18 days prior to tisagenlecleucel infusion. Within 3 days of starting lymphodepleting chemotherapy, while neutropenic, she developed severe septic shock (extended spectrum beta-lactamase-positive and ertapenem-resistant Escherichia coli) with need for broad-spectrum antibiotics, steroids, inotropes, ventilatory support, 4 days of continuous renal replacement therapy and 5 days of venoarterial extracorporeal membrane oxygenation (VA-ECMO). VA-ECMO was discontinued 11 days prior to tisagenlecleucel infusion. Shortly thereafter, the patient developed recurrent fever. A CT scan obtained for evaluation of an infectious nidus showed pulmonary opacities with tree-in-bud appearance (possible fungal etiology) and a small intra-abdominal abscess/phlegmon. Given these findings, she was started on posaconazole in addition to existing broad-spectrum antimicrobials (figure 2B). With consideration given to disease burden (11% circulating leukemic blasts on day prior to infusion), optimal antifungal/antimicrobial therapy, and stable clinical exam, the decision was made to proceed with tisagenlecleucel infusion (figure 1B). At the time of infusion, respiratory failure was resolved (without need for non-invasive ventilatory support) and the patient was hemodynamically stable. She remained on amlodipine for management of underlying hypertension, but all inotropic support was discontinued. An echocardiogram (obtained 4 days preinfusion) showed qualitatively normal right ventricular systolic function and mild global hypokinesis of the left ventricle (ejection fraction (EF) 49%, shortening fraction (SF) 32%). The patient’s postinfusion course was complicated by CRS (characterized by fever, cardiac dysfunction, fluid overload, and respiratory insufficiency) and extensive lymphadenopathy (figure 1B) with associated renovascular compromise. She received two doses of tocilizumab on day+21 given 12 hours apart with some clinical improvement, though ultimately with need for resumption of non-invasive ventilatory support and milrinone due to depressed left ventricular systolic function (EF 45%, SF 21.5%). Given the unclear nature of the patient’s aforementioned extensive adenopathy and inability to exclude disease progression, a bone marrow biopsy and aspirate were completed on day 21 postinfusion; it revealed persistent marrow disease, though notably with numerous CD3+ T lymphocytes surrounding visualized blast clusters (figure 4A–E). Follow-up biopsy and aspirate obtained 31 days postinfusion showed extensive fibrosis with no morphological or immunophenotypic evidence of disease (figure 4F). The patient was discharged from the hospital 53 days after tisagenlecleucel infusion. An echocardiogram obtained prior to discharge showed mildly depressed left ventricle systolic function (EF 49%, SF 26%) with mild left ventricular dilation. At the time of discharge (day+54 postinfusion) and on transfer home to her country of origin (day+58), she remained on amlodipine for management of hypertension but was otherwise on no cardiac remodeling medications. She remained on granulocyte colony-stimulating factor for management of severe neutropenia and was transfusion-dependent (packed red blood cells and platelets). In the setting of severe neutropenia, levofloxacin and voriconazole were also continued for infection prophylaxis. Figure 4 Bone marrow biopsy for patient 2 before and after tisagenlecleucel. (A–F) Pathology of biopsies for patient 2. Pathology of the bone marrow at 21 days (A–E) and 31 days (F) after infusion. (A, B) Blasts in the bone marrow exhibited karyorrhexis (blue arrows) mixed with intact peripheral cells 21 days after infusion. (C) CD3+ T cells surrounding and infiltrating leukemic blasts. Blue arrows indicate CD3-negative blast cells. (D) CD19 expression on the blasts. (E) Dual CD3-CD10+ immunostaining highlight T cells in brown (black arrows) and blasts in red (blue arrows). (F) Bone marrow at day 31, depleted of hematopoietic cells. The patient returned to her country of origin 58 days after infusion. Unfortunately, she expired 208 days after infusion due to cardiac arrest of unclear etiology (figure 3B). An autopsy was not obtained. Her last bone marrow evaluation was completed 3 weeks prior to death and showed no morphological or immunophenotypic evidence of disease. Discussion Here, we describe two patients with heavily pretreated r/r B-ALL who were safely infused with tisagenlecleucel despite elevated inflammatory markers, heavy leukemic burden, significant infection, and multiple comorbidities. While our center has experience with infusion of CAR-T cells dating back to 2012 and physician experience with infusions dating to 2005, tisagenlecleucel infusions are and will be the first such infusions for many centers. The complex management of patients prior to, during, and after infusion can be a source of significant concern for many providers, particularly in the presence of underlying infection, evolving disease burden, and/or other confounding variables that increase risk for severe CRS. Early recommendations to sites regarding conditions for infusion and CRS management on the pivotal multisite ELIANA trial of tisagenlecleucel were based on adult and pediatric experience at a single institution. During the trial, experienced providers were frequently consulted by those centers or providers with less experience in CAR-T cell infusion when challenged by cases for which the existing CRS management guidelines did not provide clear guidance on how to proceed (ie, a provider may inquire about tocilizumab for a patient with 5 days of persistent fever to 40°C without other signs or symptoms of CRS). Earlier in the ELIANA trial, there was significant concern that early CRS intervention might lead to diminished efficacy. Through increased patient experience in both trial and real-world settings, however, much has been learned about patient management, including management of mild to severe CRS and neurological toxicity.4 As the number of sites administering tisagenlecleucel expands and a wide range of real-world experience is obtained, it will be difficult to implement rapid changes to official guidelines. In the ELIANA trial, 43% of patients experienced infections within 8 weeks of tisagenlecleucel infusion, and 24% were severe infections (grade 3/4).2 In a few patients, prolonged severe neutropenia led to severe (grade 3 human herpesvirus 6 encephalitis) and fatal (encephalitis and systemic mycosis) infections. Analysis of real-world data in patients with B-ALL from the Center for International Blood and Marrow Transplant Research (CIBMTR) patient registry reported a similar incidence of postinfusion infections (42%), with one fatal bacterial infection.5 Furthermore, management of CRS with short-term tocilizumab therapy (IL-6 blockade) does not appear to increase the risk of infection6; however, it is important to consider that immune responses to invasive fungal infections could involve IL-67 8 and therefore may respond differently to tocilizumab therapy than was reported for our patient. Taken together, these clinical trial and real-world data highlight the known postinfusion infection risk in these patients; however, existing data fail to provide insight into how patients with preinfusion infections might be managed. Expanding the existing literature to further describe management of complex patients who, in some respects, challenge the current guidelines for infusion will be important for improving access to therapy for patients who lack other options for potential durable remission. Technically, the patients reported here were infused with tisagenlecleucel outside of label guidelines due to the presence of elevated inflammatory markers and active infection.1 For patient 1, CRS manifestations were limited to only fever without hypotension or progressive renal or pulmonary involvement. Response of B-ALL to tisagenlecleucel was not compromised by the treatment of CRS at this stage with tocilizumab. For patient 2, CRS was characterized by fever, third-spacing, cardiac dysfunction, and marked lymphadenopathy with secondary renal vascular compromise (suspected to be driven by CAR-T cell expansion). Cardiac dysfunction due to CRS has been observed in children undergoing CAR-T cell therapy, and risk of dysfunction may be increased in patients who have pre-existing cardiac abnormalities; persistent dysfunction remains uncommon, however.9 10 For patient 2, improvement in cardiac function was observed following resolution of CRS, though with unexpected death due to cardiac arrest greater than 200 days after infusion. The etiology of this arrest remains unknown at this time. Through experience gained treating CRS in patients who were part of the ELIANA study, we felt relatively comfortable that we could control CRS and therefore infused both patients under circumstances in which other providers might not have. Nevertheless, tisagenlecleucel infusion was only carried out after extensive counseling regarding the possibility of worst-case scenarios and with serious consideration also given to transport home for palliative measures. In both cases, significant discussions regarding potential life-threatening outcomes were had with patients, families, and medical providers prior to infusion. Conclusions Publication of these case studies and others may guide providers and families as they face similarly challenging circumstances in the future. In addition, they highlight the importance of clear communication with patients, families, and medical team members about potentially lethal adverse events of CAR-T cell therapy when infused in the setting of concurrent life-threatening comorbidities. While these cases represent successful infection management during CAR-T cell therapy, they should not be viewed as typical or even likely outcomes. Treatment of both patients was carried out in a setting with extensive institutional knowledge by an experienced team with the ability to customize standard protocols. Additional reporting of outcomes in patients with active infections via the CIBMTR patient registry is needed to confirm the generalizability of our approach and to better delineate benefit–risk assessment in these patients. Careful reporting on patient experiences and outcomes in the postcommercialization era of tisagenlecleucel will help to ensure safe access to potentially curative therapy for more patients. Ethics statements Patient consent for publication Obtained. Ethics approval This is a retrospective case series for which institutional review board permissions were granted and results were pulled from patients’ charts. Correction notice: This article has been corrected since it first published. The provenance and peer review statement has been included. Contributors: All authors treated patients and contributed to data collection and interpretation. EMH and GDM provided data and helped write the first draft. All authors were involved in revising the manuscript, and read and approved the final version. Funding: Medical writing support was provided by Briana Dye, PhD, of Healthcare Consultancy Group, and was funded by Novartis Pharmaceuticals Corporation. Competing interests: DEY has been an investigator on studies supported by Astellas, Chimerix, Merck, Pfizer, and Viracor-Eurofins. KJA is on the Speaker’s Bureau for and receives support from Novartis Pharmaceuticals. GDM is a consultant and is on the Speaker’s Bureau for Novartis Pharmaceuticals. EMH, RKG, AAA, GSM, SDS, TGF, IAA, WL, and RJH declare that they have no competing interests. Provenance and peer review: Not commissioned; externally peer reviewed. Supplemental material: This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.	20 MG/M2 DAILY; TO THE END OF MONTH	DrugDosageText	CC BY-NC	33472856	19,003,489	2021-01
What was the dosage of drug 'DOXORUBICIN'?	Tisagenlecleucel infusion in patients with relapsed/refractory ALL and concurrent serious infection. Tisagenlecleucel, an anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy, has demonstrated durable efficacy and a manageable safety profile in pediatric and young adult patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) in the ELIANA pivotal trial and real-world experience. Experience from investigator-led studies prior to ELIANA suggests that infections and inflammatory conditions may exacerbate the severity of cytokine release syndrome (CRS) associated with CAR-T cell therapy, leading to extreme caution and strong restrictions for on-study and commercial infusion of tisagenlecleucel in patients with active infection. CRS intervention with interleukin (IL)-6 blockade and/or steroid therapy was introduced late in the course during clinical trials due to concern for potential negative effect on efficacy and persistence. However, earlier CRS intervention is now viewed more favorably. Earlier intervention and consistency in management between providers may promote broader use of tisagenlecleucel, including potential curative therapy in patients who require remission and recovery of hematopoiesis for management of severe infection. Patient 1 was diagnosed with B-ALL at 23 years old. Fourteen days before tisagenlecleucel infusion, the patient developed fever and neutropenia and was diagnosed with invasive Mucorales infection and BK virus hemorrhagic cystitis. Aggressive measures were instituted to control infection and to manage prolonged cytopenias during CAR-T cell manufacturing. Adverse events, including CRS, were manageable despite elevated inflammatory markers and active infection. The patient attained remission and recovered hematopoiesis, and infections resolved. The patient remains in remission ≥1 year postinfusion.Patient 2 was diagnosed with pre-B-ALL at preschool age. She developed severe septic shock 3 days postinitiation of lymphodepleting chemotherapy. After receiving tisagenlecleucel, she experienced CRS with cardiac dysfunction and extensive lymphadenopathy leading to renovascular compromise. The patient attained remission and was discharged in good condition to her country of origin. She remained in remission but expired on day 208 postinfusion due to cardiac arrest of unclear etiology. Infusion was feasible, and toxicity related to tisagenlecleucel was manageable despite active infections and concurrent inflammation, allowing attainment of remission in otherwise refractory pediatric/young adult ALL. This may lead to consideration of tisagenlecleucel as a potential curative therapy in patients with managed active infections. pmcBackground Anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy (tisagenlecleucel) has demonstrated durable efficacy and manageable safety for pediatric and young adult patients with relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL).1–3 In the single-arm, phase 2 ELIANA study (NCT02435849), 79 patients with r/r B-ALL (age range, 3–24 years) were infused with a single dose of tisagenlecleucel. The overall remission rate (complete remission and complete remission with incomplete blood count recovery) was 82% within 3 months. Adverse events of special interest included cytokine release syndrome (CRS, 77%) and neurological toxicities (39%).3 Patients with active infections were not enrolled and infusion was held for cases of uncontrolled infection or inflammation. Current US prescribing information warns against administration of tisagenlecleucel to patients with active infection or inflammatory conditions.1 However, there remains a lack of data for CAR-T cell therapy in patients with active, managed infections. Furthermore, there are varying levels of comfort in treating patients with different levels of disease burden, infection types, and comorbidities. The publication of clinician and patient experiences with difficult cases should benefit providers treating patients in need of potentially curative therapy but who may be delayed or denied therapy based on prior guidelines for infusion. Herein, we describe two patients with r/r B-ALL and concurrent infection and/or organ dysfunction who were safely infused with and responded to tisagenlecleucel. Case presentation Patient 1 A 23-year-old male was diagnosed with B-ALL 18 months before tisagenlecleucel infusion. His disease relapsed after standard and salvage chemotherapy with development of refractory disease following haploidentical hematopoietic stem cell transplant. An additional text file includes further treatment history (see online supplemental file). The patient was evaluated for tisagenlecleucel and leukapheresis occurred 17 months after diagnosis. Bone marrow evaluation after bridging chemotherapy revealed 95% blasts in a normocellular marrow. 10.1136/jitc-2020-001225.supp1 Supplementary data Fourteen days preinfusion, the patient was admitted for fever/neutropenia. Due to persistent fevers, a CT scan was obtained, which demonstrated findings concerning for fungal infection. He underwent right lung thoracotomy and diaphragm biopsy and was diagnosed with invasive Mucorales infection with involvement of the right lower lung lobe, diaphragm, and liver (figure 1A). Therapy was initiated with intravenous liposomal amphotericin B, micafungin, and posaconazole (switched to isavuconazonium sulfate after 4 days); intrathoracic amphotericin B deoxycholate and granulocyte infusions were also administered. The patient remained febrile with severe pancytopenia and severe concurrent BK virus hemorrhagic cystitis. He underwent thorascopic resection of the necrotic lung by wedge resection. A posterior right mini-thoracotomy was also performed for achievement of liver debridement. Following extensive discussion regarding risk of mortality due to underlying disease progression, extensive comorbid infection, and inability to attain effective immune reconstitution in the face of heavy leukemic burden in the marrow, the decision was made to proceed with tisagenlecleucel. Lymphodepleting chemotherapy (fludarabine (dose: 30 mg/m2/day) and cytarabine (dose: 1 g/m2/day), modified due to severe lymphocytopenia and known alkylator-resistant disease) was administered over 2 days. At the time of infusion, the patient was normotensive, though still febrile and hypoxic (on supplemental nasal cannula) with three chest tubes in place following thoracotomy. The patient tolerated tisagenlecleucel infusion. Figure 1 Contrast-enhanced CT scans for both patients before tisagenlecleucel and after tisagenlecleucel infusion. (A) Patient 1: CT scans performed at diagnosis and following treatment. Axial (top left) and sagittal (bottom left) images at diagnosis demonstrate a lenticular hypodense structure (yellow arrows) centered at, and Inseparable from, the abnormally thickened right diaphragm (dashed arrows). This causes invasion or indentation of the adjacent posterior right hepatic contour. A small tubular air-filled component is present within the diaphragm (red arrows). Subtle asymmetric enlargement of the right M raises the question of chest wall involvement. There is consolidation of the adjacent right lung (). Post-treatment axial (top right) and sagittal (bottom right) images demonstrate resolution of the hypodense structure, with a residual thick-walled enhancing fluid collection along the diaphragm (yellow arrows). Note improved contour of the posterior right liver. Anastomotic sutures (red arrow) and a small left pleural effusion () are present. (B) Patient 2: CT scans performed at diagnosis and following treatment. Coronal (top left) and sagittal (bottom left) images prior to treatment demonstrate mildly enhancing soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A small amount of free fluid (*) is present. Post-treatment coronal (top right) and sagittal (bottom right) images demonstrate enlargement of the soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A, aorta; I, intrahepatic inferior vena cava; M, paraspinal muscle. Fever frequency and intensity increased several days after infusion, consistent with CRS. The patient remained normotensive with minimal oxygen requirement. On postinfusion day 8, tocilizumab was administered for a temperature of 42.9°C. Fever returned 12 hours later but eventually resolved 7 days after tocilizumab administration (figure 2A). No other signs or symptoms of CRS materialized. To help decide between palliative therapy and continued care with curative intent, an early response evaluation was performed 13 days after infusion. Bone marrow obtained at that time showed acellular morphology with 0.02% leukemia by flow cytometry. Given the low leukemic burden in the bone marrow and anticipated low likelihood of recurrent CRS, attention was turned to supporting hematopoiesis, which would be required to fully eradicate the invasive fungal infection. With this, granulocyte-macrophage colony-stimulating factor (GM-CSF) was initiated and granulocyte infusions were resumed. A repeat marrow was performed on day 28 postinfusion and showed a hypocellular marrow with no immunophenotypic evidence of disease. Due to persistent cytopenias and marrow hypocellularity, consideration was given to a CD34+ stem cell-selected boost from the patient’s mother (stem cell donor); by day+34 post-infusion, however, the patient achieved a spontaneous rise in his absolute neutrophil count. Granulocyte infusions were discontinued, and stem cell donation was canceled. The patient was discharged from the hospital 39 days after infusion. He remained on isavuconazonium, liposomal amphotericin B, and GM-CSF at time of discharge. Fifty days after infusion, GM-CSF was discontinued (figure 3A); no significant side effects were observed due to the use of GM-CSF. Figure 2 Temperature, treatment, ferritin levels, and CRP levels before and after tisagenlecleucel infusion. (A) Patient 1 temperature, treatment, ferritin levels, and CRP levels. (B) Patient 2 temperature, treatment, ferritin levels, and CRP levels. CRRT, continuous renal replacement therapy; CRP, C reactive protein; Cy, cyclophosphamide; ECMO, extracorporeal membrane oxygenation; Flu, fludarabine; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; LDC, lymphodepleting chemotherapy. Figure 3 Timeline of infections and treatment throughout the tisagenlecleucel therapy process. (A) Timeline for patient 1. aDay 8 of cefepime found muscle swelling and fungal elements in the liver; thus, the patient underwent surgery (right video-assisted thorascopic surgery with right lower lobe lung wedge resection, right thoracotomy, diaphragm biopsy, and right liver lobe wedge resection). bDiscontinued 2 days before infusion and restarted 14 days after until end of September 2018. cVideo-assisted thorascopic surgery in which a small portion of the right middle lung lobe was resected from the pleura and showed negative fungal elements. Lesion was not patched due to the potential of fungal growth. dOngoing observation. (B) Timeline for patient 2. aDelayed to December 31 due to infection. bPositive for rhinovirus/enterovirus, adenovirus, and BK virus, but remained asymptomatic. cContinued throughout blast count recovery. CRRT, continuous renal replacement therapy; GM-CSF, granulocyte-macrophage colony-stimulating factor; IVIG, intravenous immunoglobulin; LD, lymphodepleting; VA-ECMO, venoarterial extracorporeal membrane oxygenation. Forty-nine days after infusion, an additional thoracoscopic surgery was performed to resect residual necrotic lung; no fungal elements were detected at that time (figure 1A), and antifungal therapy was narrowed to monotherapy with isavuconazonium. The patient returned to his country of origin 76 days after infusion and remains in remission 19 months after infusion. Patient 2 A preschool-age Hispanic female was diagnosed with pre-B-ALL 14 months before tisagenlecleucel infusion and was treated according to the National Mexican Protocol, initially with achievement of complete remission (see online supplemental file). She developed relapsed disease 7 months prior to tisagenlecleucel infusion. She underwent reinduction chemotherapy, blinatumomab, and focal radiation; despite these therapies, bone marrow evaluation continued to show active disease 1 year after diagnosis, at which time she was referred for tisagenlecleucel infusion. The patient underwent leukapheresis 1 month prior to tisagenlecleucel infusion. She received bridging chemotherapy of 6-mercaptopurine and methotrexate and underwent lymphodepleting chemotherapy of fludarabine (30 mg/m2/dose for 3 days) and cyclophosphamide (500 mg/m2/dose for 2 days) 18 days prior to tisagenlecleucel infusion. Within 3 days of starting lymphodepleting chemotherapy, while neutropenic, she developed severe septic shock (extended spectrum beta-lactamase-positive and ertapenem-resistant Escherichia coli) with need for broad-spectrum antibiotics, steroids, inotropes, ventilatory support, 4 days of continuous renal replacement therapy and 5 days of venoarterial extracorporeal membrane oxygenation (VA-ECMO). VA-ECMO was discontinued 11 days prior to tisagenlecleucel infusion. Shortly thereafter, the patient developed recurrent fever. A CT scan obtained for evaluation of an infectious nidus showed pulmonary opacities with tree-in-bud appearance (possible fungal etiology) and a small intra-abdominal abscess/phlegmon. Given these findings, she was started on posaconazole in addition to existing broad-spectrum antimicrobials (figure 2B). With consideration given to disease burden (11% circulating leukemic blasts on day prior to infusion), optimal antifungal/antimicrobial therapy, and stable clinical exam, the decision was made to proceed with tisagenlecleucel infusion (figure 1B). At the time of infusion, respiratory failure was resolved (without need for non-invasive ventilatory support) and the patient was hemodynamically stable. She remained on amlodipine for management of underlying hypertension, but all inotropic support was discontinued. An echocardiogram (obtained 4 days preinfusion) showed qualitatively normal right ventricular systolic function and mild global hypokinesis of the left ventricle (ejection fraction (EF) 49%, shortening fraction (SF) 32%). The patient’s postinfusion course was complicated by CRS (characterized by fever, cardiac dysfunction, fluid overload, and respiratory insufficiency) and extensive lymphadenopathy (figure 1B) with associated renovascular compromise. She received two doses of tocilizumab on day+21 given 12 hours apart with some clinical improvement, though ultimately with need for resumption of non-invasive ventilatory support and milrinone due to depressed left ventricular systolic function (EF 45%, SF 21.5%). Given the unclear nature of the patient’s aforementioned extensive adenopathy and inability to exclude disease progression, a bone marrow biopsy and aspirate were completed on day 21 postinfusion; it revealed persistent marrow disease, though notably with numerous CD3+ T lymphocytes surrounding visualized blast clusters (figure 4A–E). Follow-up biopsy and aspirate obtained 31 days postinfusion showed extensive fibrosis with no morphological or immunophenotypic evidence of disease (figure 4F). The patient was discharged from the hospital 53 days after tisagenlecleucel infusion. An echocardiogram obtained prior to discharge showed mildly depressed left ventricle systolic function (EF 49%, SF 26%) with mild left ventricular dilation. At the time of discharge (day+54 postinfusion) and on transfer home to her country of origin (day+58), she remained on amlodipine for management of hypertension but was otherwise on no cardiac remodeling medications. She remained on granulocyte colony-stimulating factor for management of severe neutropenia and was transfusion-dependent (packed red blood cells and platelets). In the setting of severe neutropenia, levofloxacin and voriconazole were also continued for infection prophylaxis. Figure 4 Bone marrow biopsy for patient 2 before and after tisagenlecleucel. (A–F) Pathology of biopsies for patient 2. Pathology of the bone marrow at 21 days (A–E) and 31 days (F) after infusion. (A, B) Blasts in the bone marrow exhibited karyorrhexis (blue arrows) mixed with intact peripheral cells 21 days after infusion. (C) CD3+ T cells surrounding and infiltrating leukemic blasts. Blue arrows indicate CD3-negative blast cells. (D) CD19 expression on the blasts. (E) Dual CD3-CD10+ immunostaining highlight T cells in brown (black arrows) and blasts in red (blue arrows). (F) Bone marrow at day 31, depleted of hematopoietic cells. The patient returned to her country of origin 58 days after infusion. Unfortunately, she expired 208 days after infusion due to cardiac arrest of unclear etiology (figure 3B). An autopsy was not obtained. Her last bone marrow evaluation was completed 3 weeks prior to death and showed no morphological or immunophenotypic evidence of disease. Discussion Here, we describe two patients with heavily pretreated r/r B-ALL who were safely infused with tisagenlecleucel despite elevated inflammatory markers, heavy leukemic burden, significant infection, and multiple comorbidities. While our center has experience with infusion of CAR-T cells dating back to 2012 and physician experience with infusions dating to 2005, tisagenlecleucel infusions are and will be the first such infusions for many centers. The complex management of patients prior to, during, and after infusion can be a source of significant concern for many providers, particularly in the presence of underlying infection, evolving disease burden, and/or other confounding variables that increase risk for severe CRS. Early recommendations to sites regarding conditions for infusion and CRS management on the pivotal multisite ELIANA trial of tisagenlecleucel were based on adult and pediatric experience at a single institution. During the trial, experienced providers were frequently consulted by those centers or providers with less experience in CAR-T cell infusion when challenged by cases for which the existing CRS management guidelines did not provide clear guidance on how to proceed (ie, a provider may inquire about tocilizumab for a patient with 5 days of persistent fever to 40°C without other signs or symptoms of CRS). Earlier in the ELIANA trial, there was significant concern that early CRS intervention might lead to diminished efficacy. Through increased patient experience in both trial and real-world settings, however, much has been learned about patient management, including management of mild to severe CRS and neurological toxicity.4 As the number of sites administering tisagenlecleucel expands and a wide range of real-world experience is obtained, it will be difficult to implement rapid changes to official guidelines. In the ELIANA trial, 43% of patients experienced infections within 8 weeks of tisagenlecleucel infusion, and 24% were severe infections (grade 3/4).2 In a few patients, prolonged severe neutropenia led to severe (grade 3 human herpesvirus 6 encephalitis) and fatal (encephalitis and systemic mycosis) infections. Analysis of real-world data in patients with B-ALL from the Center for International Blood and Marrow Transplant Research (CIBMTR) patient registry reported a similar incidence of postinfusion infections (42%), with one fatal bacterial infection.5 Furthermore, management of CRS with short-term tocilizumab therapy (IL-6 blockade) does not appear to increase the risk of infection6; however, it is important to consider that immune responses to invasive fungal infections could involve IL-67 8 and therefore may respond differently to tocilizumab therapy than was reported for our patient. Taken together, these clinical trial and real-world data highlight the known postinfusion infection risk in these patients; however, existing data fail to provide insight into how patients with preinfusion infections might be managed. Expanding the existing literature to further describe management of complex patients who, in some respects, challenge the current guidelines for infusion will be important for improving access to therapy for patients who lack other options for potential durable remission. Technically, the patients reported here were infused with tisagenlecleucel outside of label guidelines due to the presence of elevated inflammatory markers and active infection.1 For patient 1, CRS manifestations were limited to only fever without hypotension or progressive renal or pulmonary involvement. Response of B-ALL to tisagenlecleucel was not compromised by the treatment of CRS at this stage with tocilizumab. For patient 2, CRS was characterized by fever, third-spacing, cardiac dysfunction, and marked lymphadenopathy with secondary renal vascular compromise (suspected to be driven by CAR-T cell expansion). Cardiac dysfunction due to CRS has been observed in children undergoing CAR-T cell therapy, and risk of dysfunction may be increased in patients who have pre-existing cardiac abnormalities; persistent dysfunction remains uncommon, however.9 10 For patient 2, improvement in cardiac function was observed following resolution of CRS, though with unexpected death due to cardiac arrest greater than 200 days after infusion. The etiology of this arrest remains unknown at this time. Through experience gained treating CRS in patients who were part of the ELIANA study, we felt relatively comfortable that we could control CRS and therefore infused both patients under circumstances in which other providers might not have. Nevertheless, tisagenlecleucel infusion was only carried out after extensive counseling regarding the possibility of worst-case scenarios and with serious consideration also given to transport home for palliative measures. In both cases, significant discussions regarding potential life-threatening outcomes were had with patients, families, and medical providers prior to infusion. Conclusions Publication of these case studies and others may guide providers and families as they face similarly challenging circumstances in the future. In addition, they highlight the importance of clear communication with patients, families, and medical team members about potentially lethal adverse events of CAR-T cell therapy when infused in the setting of concurrent life-threatening comorbidities. While these cases represent successful infection management during CAR-T cell therapy, they should not be viewed as typical or even likely outcomes. Treatment of both patients was carried out in a setting with extensive institutional knowledge by an experienced team with the ability to customize standard protocols. Additional reporting of outcomes in patients with active infections via the CIBMTR patient registry is needed to confirm the generalizability of our approach and to better delineate benefit–risk assessment in these patients. Careful reporting on patient experiences and outcomes in the postcommercialization era of tisagenlecleucel will help to ensure safe access to potentially curative therapy for more patients. Ethics statements Patient consent for publication Obtained. Ethics approval This is a retrospective case series for which institutional review board permissions were granted and results were pulled from patients’ charts. Correction notice: This article has been corrected since it first published. The provenance and peer review statement has been included. Contributors: All authors treated patients and contributed to data collection and interpretation. EMH and GDM provided data and helped write the first draft. All authors were involved in revising the manuscript, and read and approved the final version. Funding: Medical writing support was provided by Briana Dye, PhD, of Healthcare Consultancy Group, and was funded by Novartis Pharmaceuticals Corporation. Competing interests: DEY has been an investigator on studies supported by Astellas, Chimerix, Merck, Pfizer, and Viracor-Eurofins. KJA is on the Speaker’s Bureau for and receives support from Novartis Pharmaceuticals. GDM is a consultant and is on the Speaker’s Bureau for Novartis Pharmaceuticals. EMH, RKG, AAA, GSM, SDS, TGF, IAA, WL, and RJH declare that they have no competing interests. Provenance and peer review: Not commissioned; externally peer reviewed. Supplemental material: This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.	2 DOSES	DrugDosageText	CC BY-NC	33472856	19,003,489	2021-01
What was the dosage of drug 'FLUDARABINE PHOSPHATE'?	Tisagenlecleucel infusion in patients with relapsed/refractory ALL and concurrent serious infection. Tisagenlecleucel, an anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy, has demonstrated durable efficacy and a manageable safety profile in pediatric and young adult patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) in the ELIANA pivotal trial and real-world experience. Experience from investigator-led studies prior to ELIANA suggests that infections and inflammatory conditions may exacerbate the severity of cytokine release syndrome (CRS) associated with CAR-T cell therapy, leading to extreme caution and strong restrictions for on-study and commercial infusion of tisagenlecleucel in patients with active infection. CRS intervention with interleukin (IL)-6 blockade and/or steroid therapy was introduced late in the course during clinical trials due to concern for potential negative effect on efficacy and persistence. However, earlier CRS intervention is now viewed more favorably. Earlier intervention and consistency in management between providers may promote broader use of tisagenlecleucel, including potential curative therapy in patients who require remission and recovery of hematopoiesis for management of severe infection. Patient 1 was diagnosed with B-ALL at 23 years old. Fourteen days before tisagenlecleucel infusion, the patient developed fever and neutropenia and was diagnosed with invasive Mucorales infection and BK virus hemorrhagic cystitis. Aggressive measures were instituted to control infection and to manage prolonged cytopenias during CAR-T cell manufacturing. Adverse events, including CRS, were manageable despite elevated inflammatory markers and active infection. The patient attained remission and recovered hematopoiesis, and infections resolved. The patient remains in remission ≥1 year postinfusion.Patient 2 was diagnosed with pre-B-ALL at preschool age. She developed severe septic shock 3 days postinitiation of lymphodepleting chemotherapy. After receiving tisagenlecleucel, she experienced CRS with cardiac dysfunction and extensive lymphadenopathy leading to renovascular compromise. The patient attained remission and was discharged in good condition to her country of origin. She remained in remission but expired on day 208 postinfusion due to cardiac arrest of unclear etiology. Infusion was feasible, and toxicity related to tisagenlecleucel was manageable despite active infections and concurrent inflammation, allowing attainment of remission in otherwise refractory pediatric/young adult ALL. This may lead to consideration of tisagenlecleucel as a potential curative therapy in patients with managed active infections. pmcBackground Anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy (tisagenlecleucel) has demonstrated durable efficacy and manageable safety for pediatric and young adult patients with relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL).1–3 In the single-arm, phase 2 ELIANA study (NCT02435849), 79 patients with r/r B-ALL (age range, 3–24 years) were infused with a single dose of tisagenlecleucel. The overall remission rate (complete remission and complete remission with incomplete blood count recovery) was 82% within 3 months. Adverse events of special interest included cytokine release syndrome (CRS, 77%) and neurological toxicities (39%).3 Patients with active infections were not enrolled and infusion was held for cases of uncontrolled infection or inflammation. Current US prescribing information warns against administration of tisagenlecleucel to patients with active infection or inflammatory conditions.1 However, there remains a lack of data for CAR-T cell therapy in patients with active, managed infections. Furthermore, there are varying levels of comfort in treating patients with different levels of disease burden, infection types, and comorbidities. The publication of clinician and patient experiences with difficult cases should benefit providers treating patients in need of potentially curative therapy but who may be delayed or denied therapy based on prior guidelines for infusion. Herein, we describe two patients with r/r B-ALL and concurrent infection and/or organ dysfunction who were safely infused with and responded to tisagenlecleucel. Case presentation Patient 1 A 23-year-old male was diagnosed with B-ALL 18 months before tisagenlecleucel infusion. His disease relapsed after standard and salvage chemotherapy with development of refractory disease following haploidentical hematopoietic stem cell transplant. An additional text file includes further treatment history (see online supplemental file). The patient was evaluated for tisagenlecleucel and leukapheresis occurred 17 months after diagnosis. Bone marrow evaluation after bridging chemotherapy revealed 95% blasts in a normocellular marrow. 10.1136/jitc-2020-001225.supp1 Supplementary data Fourteen days preinfusion, the patient was admitted for fever/neutropenia. Due to persistent fevers, a CT scan was obtained, which demonstrated findings concerning for fungal infection. He underwent right lung thoracotomy and diaphragm biopsy and was diagnosed with invasive Mucorales infection with involvement of the right lower lung lobe, diaphragm, and liver (figure 1A). Therapy was initiated with intravenous liposomal amphotericin B, micafungin, and posaconazole (switched to isavuconazonium sulfate after 4 days); intrathoracic amphotericin B deoxycholate and granulocyte infusions were also administered. The patient remained febrile with severe pancytopenia and severe concurrent BK virus hemorrhagic cystitis. He underwent thorascopic resection of the necrotic lung by wedge resection. A posterior right mini-thoracotomy was also performed for achievement of liver debridement. Following extensive discussion regarding risk of mortality due to underlying disease progression, extensive comorbid infection, and inability to attain effective immune reconstitution in the face of heavy leukemic burden in the marrow, the decision was made to proceed with tisagenlecleucel. Lymphodepleting chemotherapy (fludarabine (dose: 30 mg/m2/day) and cytarabine (dose: 1 g/m2/day), modified due to severe lymphocytopenia and known alkylator-resistant disease) was administered over 2 days. At the time of infusion, the patient was normotensive, though still febrile and hypoxic (on supplemental nasal cannula) with three chest tubes in place following thoracotomy. The patient tolerated tisagenlecleucel infusion. Figure 1 Contrast-enhanced CT scans for both patients before tisagenlecleucel and after tisagenlecleucel infusion. (A) Patient 1: CT scans performed at diagnosis and following treatment. Axial (top left) and sagittal (bottom left) images at diagnosis demonstrate a lenticular hypodense structure (yellow arrows) centered at, and Inseparable from, the abnormally thickened right diaphragm (dashed arrows). This causes invasion or indentation of the adjacent posterior right hepatic contour. A small tubular air-filled component is present within the diaphragm (red arrows). Subtle asymmetric enlargement of the right M raises the question of chest wall involvement. There is consolidation of the adjacent right lung (). Post-treatment axial (top right) and sagittal (bottom right) images demonstrate resolution of the hypodense structure, with a residual thick-walled enhancing fluid collection along the diaphragm (yellow arrows). Note improved contour of the posterior right liver. Anastomotic sutures (red arrow) and a small left pleural effusion () are present. (B) Patient 2: CT scans performed at diagnosis and following treatment. Coronal (top left) and sagittal (bottom left) images prior to treatment demonstrate mildly enhancing soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A small amount of free fluid (*) is present. Post-treatment coronal (top right) and sagittal (bottom right) images demonstrate enlargement of the soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A, aorta; I, intrahepatic inferior vena cava; M, paraspinal muscle. Fever frequency and intensity increased several days after infusion, consistent with CRS. The patient remained normotensive with minimal oxygen requirement. On postinfusion day 8, tocilizumab was administered for a temperature of 42.9°C. Fever returned 12 hours later but eventually resolved 7 days after tocilizumab administration (figure 2A). No other signs or symptoms of CRS materialized. To help decide between palliative therapy and continued care with curative intent, an early response evaluation was performed 13 days after infusion. Bone marrow obtained at that time showed acellular morphology with 0.02% leukemia by flow cytometry. Given the low leukemic burden in the bone marrow and anticipated low likelihood of recurrent CRS, attention was turned to supporting hematopoiesis, which would be required to fully eradicate the invasive fungal infection. With this, granulocyte-macrophage colony-stimulating factor (GM-CSF) was initiated and granulocyte infusions were resumed. A repeat marrow was performed on day 28 postinfusion and showed a hypocellular marrow with no immunophenotypic evidence of disease. Due to persistent cytopenias and marrow hypocellularity, consideration was given to a CD34+ stem cell-selected boost from the patient’s mother (stem cell donor); by day+34 post-infusion, however, the patient achieved a spontaneous rise in his absolute neutrophil count. Granulocyte infusions were discontinued, and stem cell donation was canceled. The patient was discharged from the hospital 39 days after infusion. He remained on isavuconazonium, liposomal amphotericin B, and GM-CSF at time of discharge. Fifty days after infusion, GM-CSF was discontinued (figure 3A); no significant side effects were observed due to the use of GM-CSF. Figure 2 Temperature, treatment, ferritin levels, and CRP levels before and after tisagenlecleucel infusion. (A) Patient 1 temperature, treatment, ferritin levels, and CRP levels. (B) Patient 2 temperature, treatment, ferritin levels, and CRP levels. CRRT, continuous renal replacement therapy; CRP, C reactive protein; Cy, cyclophosphamide; ECMO, extracorporeal membrane oxygenation; Flu, fludarabine; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; LDC, lymphodepleting chemotherapy. Figure 3 Timeline of infections and treatment throughout the tisagenlecleucel therapy process. (A) Timeline for patient 1. aDay 8 of cefepime found muscle swelling and fungal elements in the liver; thus, the patient underwent surgery (right video-assisted thorascopic surgery with right lower lobe lung wedge resection, right thoracotomy, diaphragm biopsy, and right liver lobe wedge resection). bDiscontinued 2 days before infusion and restarted 14 days after until end of September 2018. cVideo-assisted thorascopic surgery in which a small portion of the right middle lung lobe was resected from the pleura and showed negative fungal elements. Lesion was not patched due to the potential of fungal growth. dOngoing observation. (B) Timeline for patient 2. aDelayed to December 31 due to infection. bPositive for rhinovirus/enterovirus, adenovirus, and BK virus, but remained asymptomatic. cContinued throughout blast count recovery. CRRT, continuous renal replacement therapy; GM-CSF, granulocyte-macrophage colony-stimulating factor; IVIG, intravenous immunoglobulin; LD, lymphodepleting; VA-ECMO, venoarterial extracorporeal membrane oxygenation. Forty-nine days after infusion, an additional thoracoscopic surgery was performed to resect residual necrotic lung; no fungal elements were detected at that time (figure 1A), and antifungal therapy was narrowed to monotherapy with isavuconazonium. The patient returned to his country of origin 76 days after infusion and remains in remission 19 months after infusion. Patient 2 A preschool-age Hispanic female was diagnosed with pre-B-ALL 14 months before tisagenlecleucel infusion and was treated according to the National Mexican Protocol, initially with achievement of complete remission (see online supplemental file). She developed relapsed disease 7 months prior to tisagenlecleucel infusion. She underwent reinduction chemotherapy, blinatumomab, and focal radiation; despite these therapies, bone marrow evaluation continued to show active disease 1 year after diagnosis, at which time she was referred for tisagenlecleucel infusion. The patient underwent leukapheresis 1 month prior to tisagenlecleucel infusion. She received bridging chemotherapy of 6-mercaptopurine and methotrexate and underwent lymphodepleting chemotherapy of fludarabine (30 mg/m2/dose for 3 days) and cyclophosphamide (500 mg/m2/dose for 2 days) 18 days prior to tisagenlecleucel infusion. Within 3 days of starting lymphodepleting chemotherapy, while neutropenic, she developed severe septic shock (extended spectrum beta-lactamase-positive and ertapenem-resistant Escherichia coli) with need for broad-spectrum antibiotics, steroids, inotropes, ventilatory support, 4 days of continuous renal replacement therapy and 5 days of venoarterial extracorporeal membrane oxygenation (VA-ECMO). VA-ECMO was discontinued 11 days prior to tisagenlecleucel infusion. Shortly thereafter, the patient developed recurrent fever. A CT scan obtained for evaluation of an infectious nidus showed pulmonary opacities with tree-in-bud appearance (possible fungal etiology) and a small intra-abdominal abscess/phlegmon. Given these findings, she was started on posaconazole in addition to existing broad-spectrum antimicrobials (figure 2B). With consideration given to disease burden (11% circulating leukemic blasts on day prior to infusion), optimal antifungal/antimicrobial therapy, and stable clinical exam, the decision was made to proceed with tisagenlecleucel infusion (figure 1B). At the time of infusion, respiratory failure was resolved (without need for non-invasive ventilatory support) and the patient was hemodynamically stable. She remained on amlodipine for management of underlying hypertension, but all inotropic support was discontinued. An echocardiogram (obtained 4 days preinfusion) showed qualitatively normal right ventricular systolic function and mild global hypokinesis of the left ventricle (ejection fraction (EF) 49%, shortening fraction (SF) 32%). The patient’s postinfusion course was complicated by CRS (characterized by fever, cardiac dysfunction, fluid overload, and respiratory insufficiency) and extensive lymphadenopathy (figure 1B) with associated renovascular compromise. She received two doses of tocilizumab on day+21 given 12 hours apart with some clinical improvement, though ultimately with need for resumption of non-invasive ventilatory support and milrinone due to depressed left ventricular systolic function (EF 45%, SF 21.5%). Given the unclear nature of the patient’s aforementioned extensive adenopathy and inability to exclude disease progression, a bone marrow biopsy and aspirate were completed on day 21 postinfusion; it revealed persistent marrow disease, though notably with numerous CD3+ T lymphocytes surrounding visualized blast clusters (figure 4A–E). Follow-up biopsy and aspirate obtained 31 days postinfusion showed extensive fibrosis with no morphological or immunophenotypic evidence of disease (figure 4F). The patient was discharged from the hospital 53 days after tisagenlecleucel infusion. An echocardiogram obtained prior to discharge showed mildly depressed left ventricle systolic function (EF 49%, SF 26%) with mild left ventricular dilation. At the time of discharge (day+54 postinfusion) and on transfer home to her country of origin (day+58), she remained on amlodipine for management of hypertension but was otherwise on no cardiac remodeling medications. She remained on granulocyte colony-stimulating factor for management of severe neutropenia and was transfusion-dependent (packed red blood cells and platelets). In the setting of severe neutropenia, levofloxacin and voriconazole were also continued for infection prophylaxis. Figure 4 Bone marrow biopsy for patient 2 before and after tisagenlecleucel. (A–F) Pathology of biopsies for patient 2. Pathology of the bone marrow at 21 days (A–E) and 31 days (F) after infusion. (A, B) Blasts in the bone marrow exhibited karyorrhexis (blue arrows) mixed with intact peripheral cells 21 days after infusion. (C) CD3+ T cells surrounding and infiltrating leukemic blasts. Blue arrows indicate CD3-negative blast cells. (D) CD19 expression on the blasts. (E) Dual CD3-CD10+ immunostaining highlight T cells in brown (black arrows) and blasts in red (blue arrows). (F) Bone marrow at day 31, depleted of hematopoietic cells. The patient returned to her country of origin 58 days after infusion. Unfortunately, she expired 208 days after infusion due to cardiac arrest of unclear etiology (figure 3B). An autopsy was not obtained. Her last bone marrow evaluation was completed 3 weeks prior to death and showed no morphological or immunophenotypic evidence of disease. Discussion Here, we describe two patients with heavily pretreated r/r B-ALL who were safely infused with tisagenlecleucel despite elevated inflammatory markers, heavy leukemic burden, significant infection, and multiple comorbidities. While our center has experience with infusion of CAR-T cells dating back to 2012 and physician experience with infusions dating to 2005, tisagenlecleucel infusions are and will be the first such infusions for many centers. The complex management of patients prior to, during, and after infusion can be a source of significant concern for many providers, particularly in the presence of underlying infection, evolving disease burden, and/or other confounding variables that increase risk for severe CRS. Early recommendations to sites regarding conditions for infusion and CRS management on the pivotal multisite ELIANA trial of tisagenlecleucel were based on adult and pediatric experience at a single institution. During the trial, experienced providers were frequently consulted by those centers or providers with less experience in CAR-T cell infusion when challenged by cases for which the existing CRS management guidelines did not provide clear guidance on how to proceed (ie, a provider may inquire about tocilizumab for a patient with 5 days of persistent fever to 40°C without other signs or symptoms of CRS). Earlier in the ELIANA trial, there was significant concern that early CRS intervention might lead to diminished efficacy. Through increased patient experience in both trial and real-world settings, however, much has been learned about patient management, including management of mild to severe CRS and neurological toxicity.4 As the number of sites administering tisagenlecleucel expands and a wide range of real-world experience is obtained, it will be difficult to implement rapid changes to official guidelines. In the ELIANA trial, 43% of patients experienced infections within 8 weeks of tisagenlecleucel infusion, and 24% were severe infections (grade 3/4).2 In a few patients, prolonged severe neutropenia led to severe (grade 3 human herpesvirus 6 encephalitis) and fatal (encephalitis and systemic mycosis) infections. Analysis of real-world data in patients with B-ALL from the Center for International Blood and Marrow Transplant Research (CIBMTR) patient registry reported a similar incidence of postinfusion infections (42%), with one fatal bacterial infection.5 Furthermore, management of CRS with short-term tocilizumab therapy (IL-6 blockade) does not appear to increase the risk of infection6; however, it is important to consider that immune responses to invasive fungal infections could involve IL-67 8 and therefore may respond differently to tocilizumab therapy than was reported for our patient. Taken together, these clinical trial and real-world data highlight the known postinfusion infection risk in these patients; however, existing data fail to provide insight into how patients with preinfusion infections might be managed. Expanding the existing literature to further describe management of complex patients who, in some respects, challenge the current guidelines for infusion will be important for improving access to therapy for patients who lack other options for potential durable remission. Technically, the patients reported here were infused with tisagenlecleucel outside of label guidelines due to the presence of elevated inflammatory markers and active infection.1 For patient 1, CRS manifestations were limited to only fever without hypotension or progressive renal or pulmonary involvement. Response of B-ALL to tisagenlecleucel was not compromised by the treatment of CRS at this stage with tocilizumab. For patient 2, CRS was characterized by fever, third-spacing, cardiac dysfunction, and marked lymphadenopathy with secondary renal vascular compromise (suspected to be driven by CAR-T cell expansion). Cardiac dysfunction due to CRS has been observed in children undergoing CAR-T cell therapy, and risk of dysfunction may be increased in patients who have pre-existing cardiac abnormalities; persistent dysfunction remains uncommon, however.9 10 For patient 2, improvement in cardiac function was observed following resolution of CRS, though with unexpected death due to cardiac arrest greater than 200 days after infusion. The etiology of this arrest remains unknown at this time. Through experience gained treating CRS in patients who were part of the ELIANA study, we felt relatively comfortable that we could control CRS and therefore infused both patients under circumstances in which other providers might not have. Nevertheless, tisagenlecleucel infusion was only carried out after extensive counseling regarding the possibility of worst-case scenarios and with serious consideration also given to transport home for palliative measures. In both cases, significant discussions regarding potential life-threatening outcomes were had with patients, families, and medical providers prior to infusion. Conclusions Publication of these case studies and others may guide providers and families as they face similarly challenging circumstances in the future. In addition, they highlight the importance of clear communication with patients, families, and medical team members about potentially lethal adverse events of CAR-T cell therapy when infused in the setting of concurrent life-threatening comorbidities. While these cases represent successful infection management during CAR-T cell therapy, they should not be viewed as typical or even likely outcomes. Treatment of both patients was carried out in a setting with extensive institutional knowledge by an experienced team with the ability to customize standard protocols. Additional reporting of outcomes in patients with active infections via the CIBMTR patient registry is needed to confirm the generalizability of our approach and to better delineate benefit–risk assessment in these patients. Careful reporting on patient experiences and outcomes in the postcommercialization era of tisagenlecleucel will help to ensure safe access to potentially curative therapy for more patients. Ethics statements Patient consent for publication Obtained. Ethics approval This is a retrospective case series for which institutional review board permissions were granted and results were pulled from patients’ charts. Correction notice: This article has been corrected since it first published. The provenance and peer review statement has been included. Contributors: All authors treated patients and contributed to data collection and interpretation. EMH and GDM provided data and helped write the first draft. All authors were involved in revising the manuscript, and read and approved the final version. Funding: Medical writing support was provided by Briana Dye, PhD, of Healthcare Consultancy Group, and was funded by Novartis Pharmaceuticals Corporation. Competing interests: DEY has been an investigator on studies supported by Astellas, Chimerix, Merck, Pfizer, and Viracor-Eurofins. KJA is on the Speaker’s Bureau for and receives support from Novartis Pharmaceuticals. GDM is a consultant and is on the Speaker’s Bureau for Novartis Pharmaceuticals. EMH, RKG, AAA, GSM, SDS, TGF, IAA, WL, and RJH declare that they have no competing interests. Provenance and peer review: Not commissioned; externally peer reviewed. Supplemental material: This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.	3 MG/M2 PER DOSE	DrugDosageText	CC BY-NC	33472856	19,003,489	2021-01
What was the dosage of drug 'PEGASPARGASE'?	Tisagenlecleucel infusion in patients with relapsed/refractory ALL and concurrent serious infection. Tisagenlecleucel, an anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy, has demonstrated durable efficacy and a manageable safety profile in pediatric and young adult patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) in the ELIANA pivotal trial and real-world experience. Experience from investigator-led studies prior to ELIANA suggests that infections and inflammatory conditions may exacerbate the severity of cytokine release syndrome (CRS) associated with CAR-T cell therapy, leading to extreme caution and strong restrictions for on-study and commercial infusion of tisagenlecleucel in patients with active infection. CRS intervention with interleukin (IL)-6 blockade and/or steroid therapy was introduced late in the course during clinical trials due to concern for potential negative effect on efficacy and persistence. However, earlier CRS intervention is now viewed more favorably. Earlier intervention and consistency in management between providers may promote broader use of tisagenlecleucel, including potential curative therapy in patients who require remission and recovery of hematopoiesis for management of severe infection. Patient 1 was diagnosed with B-ALL at 23 years old. Fourteen days before tisagenlecleucel infusion, the patient developed fever and neutropenia and was diagnosed with invasive Mucorales infection and BK virus hemorrhagic cystitis. Aggressive measures were instituted to control infection and to manage prolonged cytopenias during CAR-T cell manufacturing. Adverse events, including CRS, were manageable despite elevated inflammatory markers and active infection. The patient attained remission and recovered hematopoiesis, and infections resolved. The patient remains in remission ≥1 year postinfusion.Patient 2 was diagnosed with pre-B-ALL at preschool age. She developed severe septic shock 3 days postinitiation of lymphodepleting chemotherapy. After receiving tisagenlecleucel, she experienced CRS with cardiac dysfunction and extensive lymphadenopathy leading to renovascular compromise. The patient attained remission and was discharged in good condition to her country of origin. She remained in remission but expired on day 208 postinfusion due to cardiac arrest of unclear etiology. Infusion was feasible, and toxicity related to tisagenlecleucel was manageable despite active infections and concurrent inflammation, allowing attainment of remission in otherwise refractory pediatric/young adult ALL. This may lead to consideration of tisagenlecleucel as a potential curative therapy in patients with managed active infections. pmcBackground Anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy (tisagenlecleucel) has demonstrated durable efficacy and manageable safety for pediatric and young adult patients with relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL).1–3 In the single-arm, phase 2 ELIANA study (NCT02435849), 79 patients with r/r B-ALL (age range, 3–24 years) were infused with a single dose of tisagenlecleucel. The overall remission rate (complete remission and complete remission with incomplete blood count recovery) was 82% within 3 months. Adverse events of special interest included cytokine release syndrome (CRS, 77%) and neurological toxicities (39%).3 Patients with active infections were not enrolled and infusion was held for cases of uncontrolled infection or inflammation. Current US prescribing information warns against administration of tisagenlecleucel to patients with active infection or inflammatory conditions.1 However, there remains a lack of data for CAR-T cell therapy in patients with active, managed infections. Furthermore, there are varying levels of comfort in treating patients with different levels of disease burden, infection types, and comorbidities. The publication of clinician and patient experiences with difficult cases should benefit providers treating patients in need of potentially curative therapy but who may be delayed or denied therapy based on prior guidelines for infusion. Herein, we describe two patients with r/r B-ALL and concurrent infection and/or organ dysfunction who were safely infused with and responded to tisagenlecleucel. Case presentation Patient 1 A 23-year-old male was diagnosed with B-ALL 18 months before tisagenlecleucel infusion. His disease relapsed after standard and salvage chemotherapy with development of refractory disease following haploidentical hematopoietic stem cell transplant. An additional text file includes further treatment history (see online supplemental file). The patient was evaluated for tisagenlecleucel and leukapheresis occurred 17 months after diagnosis. Bone marrow evaluation after bridging chemotherapy revealed 95% blasts in a normocellular marrow. 10.1136/jitc-2020-001225.supp1 Supplementary data Fourteen days preinfusion, the patient was admitted for fever/neutropenia. Due to persistent fevers, a CT scan was obtained, which demonstrated findings concerning for fungal infection. He underwent right lung thoracotomy and diaphragm biopsy and was diagnosed with invasive Mucorales infection with involvement of the right lower lung lobe, diaphragm, and liver (figure 1A). Therapy was initiated with intravenous liposomal amphotericin B, micafungin, and posaconazole (switched to isavuconazonium sulfate after 4 days); intrathoracic amphotericin B deoxycholate and granulocyte infusions were also administered. The patient remained febrile with severe pancytopenia and severe concurrent BK virus hemorrhagic cystitis. He underwent thorascopic resection of the necrotic lung by wedge resection. A posterior right mini-thoracotomy was also performed for achievement of liver debridement. Following extensive discussion regarding risk of mortality due to underlying disease progression, extensive comorbid infection, and inability to attain effective immune reconstitution in the face of heavy leukemic burden in the marrow, the decision was made to proceed with tisagenlecleucel. Lymphodepleting chemotherapy (fludarabine (dose: 30 mg/m2/day) and cytarabine (dose: 1 g/m2/day), modified due to severe lymphocytopenia and known alkylator-resistant disease) was administered over 2 days. At the time of infusion, the patient was normotensive, though still febrile and hypoxic (on supplemental nasal cannula) with three chest tubes in place following thoracotomy. The patient tolerated tisagenlecleucel infusion. Figure 1 Contrast-enhanced CT scans for both patients before tisagenlecleucel and after tisagenlecleucel infusion. (A) Patient 1: CT scans performed at diagnosis and following treatment. Axial (top left) and sagittal (bottom left) images at diagnosis demonstrate a lenticular hypodense structure (yellow arrows) centered at, and Inseparable from, the abnormally thickened right diaphragm (dashed arrows). This causes invasion or indentation of the adjacent posterior right hepatic contour. A small tubular air-filled component is present within the diaphragm (red arrows). Subtle asymmetric enlargement of the right M raises the question of chest wall involvement. There is consolidation of the adjacent right lung (). Post-treatment axial (top right) and sagittal (bottom right) images demonstrate resolution of the hypodense structure, with a residual thick-walled enhancing fluid collection along the diaphragm (yellow arrows). Note improved contour of the posterior right liver. Anastomotic sutures (red arrow) and a small left pleural effusion () are present. (B) Patient 2: CT scans performed at diagnosis and following treatment. Coronal (top left) and sagittal (bottom left) images prior to treatment demonstrate mildly enhancing soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A small amount of free fluid (*) is present. Post-treatment coronal (top right) and sagittal (bottom right) images demonstrate enlargement of the soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A, aorta; I, intrahepatic inferior vena cava; M, paraspinal muscle. Fever frequency and intensity increased several days after infusion, consistent with CRS. The patient remained normotensive with minimal oxygen requirement. On postinfusion day 8, tocilizumab was administered for a temperature of 42.9°C. Fever returned 12 hours later but eventually resolved 7 days after tocilizumab administration (figure 2A). No other signs or symptoms of CRS materialized. To help decide between palliative therapy and continued care with curative intent, an early response evaluation was performed 13 days after infusion. Bone marrow obtained at that time showed acellular morphology with 0.02% leukemia by flow cytometry. Given the low leukemic burden in the bone marrow and anticipated low likelihood of recurrent CRS, attention was turned to supporting hematopoiesis, which would be required to fully eradicate the invasive fungal infection. With this, granulocyte-macrophage colony-stimulating factor (GM-CSF) was initiated and granulocyte infusions were resumed. A repeat marrow was performed on day 28 postinfusion and showed a hypocellular marrow with no immunophenotypic evidence of disease. Due to persistent cytopenias and marrow hypocellularity, consideration was given to a CD34+ stem cell-selected boost from the patient’s mother (stem cell donor); by day+34 post-infusion, however, the patient achieved a spontaneous rise in his absolute neutrophil count. Granulocyte infusions were discontinued, and stem cell donation was canceled. The patient was discharged from the hospital 39 days after infusion. He remained on isavuconazonium, liposomal amphotericin B, and GM-CSF at time of discharge. Fifty days after infusion, GM-CSF was discontinued (figure 3A); no significant side effects were observed due to the use of GM-CSF. Figure 2 Temperature, treatment, ferritin levels, and CRP levels before and after tisagenlecleucel infusion. (A) Patient 1 temperature, treatment, ferritin levels, and CRP levels. (B) Patient 2 temperature, treatment, ferritin levels, and CRP levels. CRRT, continuous renal replacement therapy; CRP, C reactive protein; Cy, cyclophosphamide; ECMO, extracorporeal membrane oxygenation; Flu, fludarabine; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; LDC, lymphodepleting chemotherapy. Figure 3 Timeline of infections and treatment throughout the tisagenlecleucel therapy process. (A) Timeline for patient 1. aDay 8 of cefepime found muscle swelling and fungal elements in the liver; thus, the patient underwent surgery (right video-assisted thorascopic surgery with right lower lobe lung wedge resection, right thoracotomy, diaphragm biopsy, and right liver lobe wedge resection). bDiscontinued 2 days before infusion and restarted 14 days after until end of September 2018. cVideo-assisted thorascopic surgery in which a small portion of the right middle lung lobe was resected from the pleura and showed negative fungal elements. Lesion was not patched due to the potential of fungal growth. dOngoing observation. (B) Timeline for patient 2. aDelayed to December 31 due to infection. bPositive for rhinovirus/enterovirus, adenovirus, and BK virus, but remained asymptomatic. cContinued throughout blast count recovery. CRRT, continuous renal replacement therapy; GM-CSF, granulocyte-macrophage colony-stimulating factor; IVIG, intravenous immunoglobulin; LD, lymphodepleting; VA-ECMO, venoarterial extracorporeal membrane oxygenation. Forty-nine days after infusion, an additional thoracoscopic surgery was performed to resect residual necrotic lung; no fungal elements were detected at that time (figure 1A), and antifungal therapy was narrowed to monotherapy with isavuconazonium. The patient returned to his country of origin 76 days after infusion and remains in remission 19 months after infusion. Patient 2 A preschool-age Hispanic female was diagnosed with pre-B-ALL 14 months before tisagenlecleucel infusion and was treated according to the National Mexican Protocol, initially with achievement of complete remission (see online supplemental file). She developed relapsed disease 7 months prior to tisagenlecleucel infusion. She underwent reinduction chemotherapy, blinatumomab, and focal radiation; despite these therapies, bone marrow evaluation continued to show active disease 1 year after diagnosis, at which time she was referred for tisagenlecleucel infusion. The patient underwent leukapheresis 1 month prior to tisagenlecleucel infusion. She received bridging chemotherapy of 6-mercaptopurine and methotrexate and underwent lymphodepleting chemotherapy of fludarabine (30 mg/m2/dose for 3 days) and cyclophosphamide (500 mg/m2/dose for 2 days) 18 days prior to tisagenlecleucel infusion. Within 3 days of starting lymphodepleting chemotherapy, while neutropenic, she developed severe septic shock (extended spectrum beta-lactamase-positive and ertapenem-resistant Escherichia coli) with need for broad-spectrum antibiotics, steroids, inotropes, ventilatory support, 4 days of continuous renal replacement therapy and 5 days of venoarterial extracorporeal membrane oxygenation (VA-ECMO). VA-ECMO was discontinued 11 days prior to tisagenlecleucel infusion. Shortly thereafter, the patient developed recurrent fever. A CT scan obtained for evaluation of an infectious nidus showed pulmonary opacities with tree-in-bud appearance (possible fungal etiology) and a small intra-abdominal abscess/phlegmon. Given these findings, she was started on posaconazole in addition to existing broad-spectrum antimicrobials (figure 2B). With consideration given to disease burden (11% circulating leukemic blasts on day prior to infusion), optimal antifungal/antimicrobial therapy, and stable clinical exam, the decision was made to proceed with tisagenlecleucel infusion (figure 1B). At the time of infusion, respiratory failure was resolved (without need for non-invasive ventilatory support) and the patient was hemodynamically stable. She remained on amlodipine for management of underlying hypertension, but all inotropic support was discontinued. An echocardiogram (obtained 4 days preinfusion) showed qualitatively normal right ventricular systolic function and mild global hypokinesis of the left ventricle (ejection fraction (EF) 49%, shortening fraction (SF) 32%). The patient’s postinfusion course was complicated by CRS (characterized by fever, cardiac dysfunction, fluid overload, and respiratory insufficiency) and extensive lymphadenopathy (figure 1B) with associated renovascular compromise. She received two doses of tocilizumab on day+21 given 12 hours apart with some clinical improvement, though ultimately with need for resumption of non-invasive ventilatory support and milrinone due to depressed left ventricular systolic function (EF 45%, SF 21.5%). Given the unclear nature of the patient’s aforementioned extensive adenopathy and inability to exclude disease progression, a bone marrow biopsy and aspirate were completed on day 21 postinfusion; it revealed persistent marrow disease, though notably with numerous CD3+ T lymphocytes surrounding visualized blast clusters (figure 4A–E). Follow-up biopsy and aspirate obtained 31 days postinfusion showed extensive fibrosis with no morphological or immunophenotypic evidence of disease (figure 4F). The patient was discharged from the hospital 53 days after tisagenlecleucel infusion. An echocardiogram obtained prior to discharge showed mildly depressed left ventricle systolic function (EF 49%, SF 26%) with mild left ventricular dilation. At the time of discharge (day+54 postinfusion) and on transfer home to her country of origin (day+58), she remained on amlodipine for management of hypertension but was otherwise on no cardiac remodeling medications. She remained on granulocyte colony-stimulating factor for management of severe neutropenia and was transfusion-dependent (packed red blood cells and platelets). In the setting of severe neutropenia, levofloxacin and voriconazole were also continued for infection prophylaxis. Figure 4 Bone marrow biopsy for patient 2 before and after tisagenlecleucel. (A–F) Pathology of biopsies for patient 2. Pathology of the bone marrow at 21 days (A–E) and 31 days (F) after infusion. (A, B) Blasts in the bone marrow exhibited karyorrhexis (blue arrows) mixed with intact peripheral cells 21 days after infusion. (C) CD3+ T cells surrounding and infiltrating leukemic blasts. Blue arrows indicate CD3-negative blast cells. (D) CD19 expression on the blasts. (E) Dual CD3-CD10+ immunostaining highlight T cells in brown (black arrows) and blasts in red (blue arrows). (F) Bone marrow at day 31, depleted of hematopoietic cells. The patient returned to her country of origin 58 days after infusion. Unfortunately, she expired 208 days after infusion due to cardiac arrest of unclear etiology (figure 3B). An autopsy was not obtained. Her last bone marrow evaluation was completed 3 weeks prior to death and showed no morphological or immunophenotypic evidence of disease. Discussion Here, we describe two patients with heavily pretreated r/r B-ALL who were safely infused with tisagenlecleucel despite elevated inflammatory markers, heavy leukemic burden, significant infection, and multiple comorbidities. While our center has experience with infusion of CAR-T cells dating back to 2012 and physician experience with infusions dating to 2005, tisagenlecleucel infusions are and will be the first such infusions for many centers. The complex management of patients prior to, during, and after infusion can be a source of significant concern for many providers, particularly in the presence of underlying infection, evolving disease burden, and/or other confounding variables that increase risk for severe CRS. Early recommendations to sites regarding conditions for infusion and CRS management on the pivotal multisite ELIANA trial of tisagenlecleucel were based on adult and pediatric experience at a single institution. During the trial, experienced providers were frequently consulted by those centers or providers with less experience in CAR-T cell infusion when challenged by cases for which the existing CRS management guidelines did not provide clear guidance on how to proceed (ie, a provider may inquire about tocilizumab for a patient with 5 days of persistent fever to 40°C without other signs or symptoms of CRS). Earlier in the ELIANA trial, there was significant concern that early CRS intervention might lead to diminished efficacy. Through increased patient experience in both trial and real-world settings, however, much has been learned about patient management, including management of mild to severe CRS and neurological toxicity.4 As the number of sites administering tisagenlecleucel expands and a wide range of real-world experience is obtained, it will be difficult to implement rapid changes to official guidelines. In the ELIANA trial, 43% of patients experienced infections within 8 weeks of tisagenlecleucel infusion, and 24% were severe infections (grade 3/4).2 In a few patients, prolonged severe neutropenia led to severe (grade 3 human herpesvirus 6 encephalitis) and fatal (encephalitis and systemic mycosis) infections. Analysis of real-world data in patients with B-ALL from the Center for International Blood and Marrow Transplant Research (CIBMTR) patient registry reported a similar incidence of postinfusion infections (42%), with one fatal bacterial infection.5 Furthermore, management of CRS with short-term tocilizumab therapy (IL-6 blockade) does not appear to increase the risk of infection6; however, it is important to consider that immune responses to invasive fungal infections could involve IL-67 8 and therefore may respond differently to tocilizumab therapy than was reported for our patient. Taken together, these clinical trial and real-world data highlight the known postinfusion infection risk in these patients; however, existing data fail to provide insight into how patients with preinfusion infections might be managed. Expanding the existing literature to further describe management of complex patients who, in some respects, challenge the current guidelines for infusion will be important for improving access to therapy for patients who lack other options for potential durable remission. Technically, the patients reported here were infused with tisagenlecleucel outside of label guidelines due to the presence of elevated inflammatory markers and active infection.1 For patient 1, CRS manifestations were limited to only fever without hypotension or progressive renal or pulmonary involvement. Response of B-ALL to tisagenlecleucel was not compromised by the treatment of CRS at this stage with tocilizumab. For patient 2, CRS was characterized by fever, third-spacing, cardiac dysfunction, and marked lymphadenopathy with secondary renal vascular compromise (suspected to be driven by CAR-T cell expansion). Cardiac dysfunction due to CRS has been observed in children undergoing CAR-T cell therapy, and risk of dysfunction may be increased in patients who have pre-existing cardiac abnormalities; persistent dysfunction remains uncommon, however.9 10 For patient 2, improvement in cardiac function was observed following resolution of CRS, though with unexpected death due to cardiac arrest greater than 200 days after infusion. The etiology of this arrest remains unknown at this time. Through experience gained treating CRS in patients who were part of the ELIANA study, we felt relatively comfortable that we could control CRS and therefore infused both patients under circumstances in which other providers might not have. Nevertheless, tisagenlecleucel infusion was only carried out after extensive counseling regarding the possibility of worst-case scenarios and with serious consideration also given to transport home for palliative measures. In both cases, significant discussions regarding potential life-threatening outcomes were had with patients, families, and medical providers prior to infusion. Conclusions Publication of these case studies and others may guide providers and families as they face similarly challenging circumstances in the future. In addition, they highlight the importance of clear communication with patients, families, and medical team members about potentially lethal adverse events of CAR-T cell therapy when infused in the setting of concurrent life-threatening comorbidities. While these cases represent successful infection management during CAR-T cell therapy, they should not be viewed as typical or even likely outcomes. Treatment of both patients was carried out in a setting with extensive institutional knowledge by an experienced team with the ability to customize standard protocols. Additional reporting of outcomes in patients with active infections via the CIBMTR patient registry is needed to confirm the generalizability of our approach and to better delineate benefit–risk assessment in these patients. Careful reporting on patient experiences and outcomes in the postcommercialization era of tisagenlecleucel will help to ensure safe access to potentially curative therapy for more patients. Ethics statements Patient consent for publication Obtained. Ethics approval This is a retrospective case series for which institutional review board permissions were granted and results were pulled from patients’ charts. Correction notice: This article has been corrected since it first published. The provenance and peer review statement has been included. Contributors: All authors treated patients and contributed to data collection and interpretation. EMH and GDM provided data and helped write the first draft. All authors were involved in revising the manuscript, and read and approved the final version. Funding: Medical writing support was provided by Briana Dye, PhD, of Healthcare Consultancy Group, and was funded by Novartis Pharmaceuticals Corporation. Competing interests: DEY has been an investigator on studies supported by Astellas, Chimerix, Merck, Pfizer, and Viracor-Eurofins. KJA is on the Speaker’s Bureau for and receives support from Novartis Pharmaceuticals. GDM is a consultant and is on the Speaker’s Bureau for Novartis Pharmaceuticals. EMH, RKG, AAA, GSM, SDS, TGF, IAA, WL, and RJH declare that they have no competing interests. Provenance and peer review: Not commissioned; externally peer reviewed. Supplemental material: This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.	1000 IU/M2 ON DAYS 3 AND 15 FOR 2 MONTHS	DrugDosageText	CC BY-NC	33472856	19,003,489	2021-01
What was the dosage of drug 'PREDNISONE'?	Tisagenlecleucel infusion in patients with relapsed/refractory ALL and concurrent serious infection. Tisagenlecleucel, an anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy, has demonstrated durable efficacy and a manageable safety profile in pediatric and young adult patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) in the ELIANA pivotal trial and real-world experience. Experience from investigator-led studies prior to ELIANA suggests that infections and inflammatory conditions may exacerbate the severity of cytokine release syndrome (CRS) associated with CAR-T cell therapy, leading to extreme caution and strong restrictions for on-study and commercial infusion of tisagenlecleucel in patients with active infection. CRS intervention with interleukin (IL)-6 blockade and/or steroid therapy was introduced late in the course during clinical trials due to concern for potential negative effect on efficacy and persistence. However, earlier CRS intervention is now viewed more favorably. Earlier intervention and consistency in management between providers may promote broader use of tisagenlecleucel, including potential curative therapy in patients who require remission and recovery of hematopoiesis for management of severe infection. Patient 1 was diagnosed with B-ALL at 23 years old. Fourteen days before tisagenlecleucel infusion, the patient developed fever and neutropenia and was diagnosed with invasive Mucorales infection and BK virus hemorrhagic cystitis. Aggressive measures were instituted to control infection and to manage prolonged cytopenias during CAR-T cell manufacturing. Adverse events, including CRS, were manageable despite elevated inflammatory markers and active infection. The patient attained remission and recovered hematopoiesis, and infections resolved. The patient remains in remission ≥1 year postinfusion.Patient 2 was diagnosed with pre-B-ALL at preschool age. She developed severe septic shock 3 days postinitiation of lymphodepleting chemotherapy. After receiving tisagenlecleucel, she experienced CRS with cardiac dysfunction and extensive lymphadenopathy leading to renovascular compromise. The patient attained remission and was discharged in good condition to her country of origin. She remained in remission but expired on day 208 postinfusion due to cardiac arrest of unclear etiology. Infusion was feasible, and toxicity related to tisagenlecleucel was manageable despite active infections and concurrent inflammation, allowing attainment of remission in otherwise refractory pediatric/young adult ALL. This may lead to consideration of tisagenlecleucel as a potential curative therapy in patients with managed active infections. pmcBackground Anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy (tisagenlecleucel) has demonstrated durable efficacy and manageable safety for pediatric and young adult patients with relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL).1–3 In the single-arm, phase 2 ELIANA study (NCT02435849), 79 patients with r/r B-ALL (age range, 3–24 years) were infused with a single dose of tisagenlecleucel. The overall remission rate (complete remission and complete remission with incomplete blood count recovery) was 82% within 3 months. Adverse events of special interest included cytokine release syndrome (CRS, 77%) and neurological toxicities (39%).3 Patients with active infections were not enrolled and infusion was held for cases of uncontrolled infection or inflammation. Current US prescribing information warns against administration of tisagenlecleucel to patients with active infection or inflammatory conditions.1 However, there remains a lack of data for CAR-T cell therapy in patients with active, managed infections. Furthermore, there are varying levels of comfort in treating patients with different levels of disease burden, infection types, and comorbidities. The publication of clinician and patient experiences with difficult cases should benefit providers treating patients in need of potentially curative therapy but who may be delayed or denied therapy based on prior guidelines for infusion. Herein, we describe two patients with r/r B-ALL and concurrent infection and/or organ dysfunction who were safely infused with and responded to tisagenlecleucel. Case presentation Patient 1 A 23-year-old male was diagnosed with B-ALL 18 months before tisagenlecleucel infusion. His disease relapsed after standard and salvage chemotherapy with development of refractory disease following haploidentical hematopoietic stem cell transplant. An additional text file includes further treatment history (see online supplemental file). The patient was evaluated for tisagenlecleucel and leukapheresis occurred 17 months after diagnosis. Bone marrow evaluation after bridging chemotherapy revealed 95% blasts in a normocellular marrow. 10.1136/jitc-2020-001225.supp1 Supplementary data Fourteen days preinfusion, the patient was admitted for fever/neutropenia. Due to persistent fevers, a CT scan was obtained, which demonstrated findings concerning for fungal infection. He underwent right lung thoracotomy and diaphragm biopsy and was diagnosed with invasive Mucorales infection with involvement of the right lower lung lobe, diaphragm, and liver (figure 1A). Therapy was initiated with intravenous liposomal amphotericin B, micafungin, and posaconazole (switched to isavuconazonium sulfate after 4 days); intrathoracic amphotericin B deoxycholate and granulocyte infusions were also administered. The patient remained febrile with severe pancytopenia and severe concurrent BK virus hemorrhagic cystitis. He underwent thorascopic resection of the necrotic lung by wedge resection. A posterior right mini-thoracotomy was also performed for achievement of liver debridement. Following extensive discussion regarding risk of mortality due to underlying disease progression, extensive comorbid infection, and inability to attain effective immune reconstitution in the face of heavy leukemic burden in the marrow, the decision was made to proceed with tisagenlecleucel. Lymphodepleting chemotherapy (fludarabine (dose: 30 mg/m2/day) and cytarabine (dose: 1 g/m2/day), modified due to severe lymphocytopenia and known alkylator-resistant disease) was administered over 2 days. At the time of infusion, the patient was normotensive, though still febrile and hypoxic (on supplemental nasal cannula) with three chest tubes in place following thoracotomy. The patient tolerated tisagenlecleucel infusion. Figure 1 Contrast-enhanced CT scans for both patients before tisagenlecleucel and after tisagenlecleucel infusion. (A) Patient 1: CT scans performed at diagnosis and following treatment. Axial (top left) and sagittal (bottom left) images at diagnosis demonstrate a lenticular hypodense structure (yellow arrows) centered at, and Inseparable from, the abnormally thickened right diaphragm (dashed arrows). This causes invasion or indentation of the adjacent posterior right hepatic contour. A small tubular air-filled component is present within the diaphragm (red arrows). Subtle asymmetric enlargement of the right M raises the question of chest wall involvement. There is consolidation of the adjacent right lung (). Post-treatment axial (top right) and sagittal (bottom right) images demonstrate resolution of the hypodense structure, with a residual thick-walled enhancing fluid collection along the diaphragm (yellow arrows). Note improved contour of the posterior right liver. Anastomotic sutures (red arrow) and a small left pleural effusion () are present. (B) Patient 2: CT scans performed at diagnosis and following treatment. Coronal (top left) and sagittal (bottom left) images prior to treatment demonstrate mildly enhancing soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A small amount of free fluid (*) is present. Post-treatment coronal (top right) and sagittal (bottom right) images demonstrate enlargement of the soft tissue nodal masses (large yellow arrows) encircling the renal arteries (red arrows) and veins (small dashed arrows). A and I are partially visualized. A, aorta; I, intrahepatic inferior vena cava; M, paraspinal muscle. Fever frequency and intensity increased several days after infusion, consistent with CRS. The patient remained normotensive with minimal oxygen requirement. On postinfusion day 8, tocilizumab was administered for a temperature of 42.9°C. Fever returned 12 hours later but eventually resolved 7 days after tocilizumab administration (figure 2A). No other signs or symptoms of CRS materialized. To help decide between palliative therapy and continued care with curative intent, an early response evaluation was performed 13 days after infusion. Bone marrow obtained at that time showed acellular morphology with 0.02% leukemia by flow cytometry. Given the low leukemic burden in the bone marrow and anticipated low likelihood of recurrent CRS, attention was turned to supporting hematopoiesis, which would be required to fully eradicate the invasive fungal infection. With this, granulocyte-macrophage colony-stimulating factor (GM-CSF) was initiated and granulocyte infusions were resumed. A repeat marrow was performed on day 28 postinfusion and showed a hypocellular marrow with no immunophenotypic evidence of disease. Due to persistent cytopenias and marrow hypocellularity, consideration was given to a CD34+ stem cell-selected boost from the patient’s mother (stem cell donor); by day+34 post-infusion, however, the patient achieved a spontaneous rise in his absolute neutrophil count. Granulocyte infusions were discontinued, and stem cell donation was canceled. The patient was discharged from the hospital 39 days after infusion. He remained on isavuconazonium, liposomal amphotericin B, and GM-CSF at time of discharge. Fifty days after infusion, GM-CSF was discontinued (figure 3A); no significant side effects were observed due to the use of GM-CSF. Figure 2 Temperature, treatment, ferritin levels, and CRP levels before and after tisagenlecleucel infusion. (A) Patient 1 temperature, treatment, ferritin levels, and CRP levels. (B) Patient 2 temperature, treatment, ferritin levels, and CRP levels. CRRT, continuous renal replacement therapy; CRP, C reactive protein; Cy, cyclophosphamide; ECMO, extracorporeal membrane oxygenation; Flu, fludarabine; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; LDC, lymphodepleting chemotherapy. Figure 3 Timeline of infections and treatment throughout the tisagenlecleucel therapy process. (A) Timeline for patient 1. aDay 8 of cefepime found muscle swelling and fungal elements in the liver; thus, the patient underwent surgery (right video-assisted thorascopic surgery with right lower lobe lung wedge resection, right thoracotomy, diaphragm biopsy, and right liver lobe wedge resection). bDiscontinued 2 days before infusion and restarted 14 days after until end of September 2018. cVideo-assisted thorascopic surgery in which a small portion of the right middle lung lobe was resected from the pleura and showed negative fungal elements. Lesion was not patched due to the potential of fungal growth. dOngoing observation. (B) Timeline for patient 2. aDelayed to December 31 due to infection. bPositive for rhinovirus/enterovirus, adenovirus, and BK virus, but remained asymptomatic. cContinued throughout blast count recovery. CRRT, continuous renal replacement therapy; GM-CSF, granulocyte-macrophage colony-stimulating factor; IVIG, intravenous immunoglobulin; LD, lymphodepleting; VA-ECMO, venoarterial extracorporeal membrane oxygenation. Forty-nine days after infusion, an additional thoracoscopic surgery was performed to resect residual necrotic lung; no fungal elements were detected at that time (figure 1A), and antifungal therapy was narrowed to monotherapy with isavuconazonium. The patient returned to his country of origin 76 days after infusion and remains in remission 19 months after infusion. Patient 2 A preschool-age Hispanic female was diagnosed with pre-B-ALL 14 months before tisagenlecleucel infusion and was treated according to the National Mexican Protocol, initially with achievement of complete remission (see online supplemental file). She developed relapsed disease 7 months prior to tisagenlecleucel infusion. She underwent reinduction chemotherapy, blinatumomab, and focal radiation; despite these therapies, bone marrow evaluation continued to show active disease 1 year after diagnosis, at which time she was referred for tisagenlecleucel infusion. The patient underwent leukapheresis 1 month prior to tisagenlecleucel infusion. She received bridging chemotherapy of 6-mercaptopurine and methotrexate and underwent lymphodepleting chemotherapy of fludarabine (30 mg/m2/dose for 3 days) and cyclophosphamide (500 mg/m2/dose for 2 days) 18 days prior to tisagenlecleucel infusion. Within 3 days of starting lymphodepleting chemotherapy, while neutropenic, she developed severe septic shock (extended spectrum beta-lactamase-positive and ertapenem-resistant Escherichia coli) with need for broad-spectrum antibiotics, steroids, inotropes, ventilatory support, 4 days of continuous renal replacement therapy and 5 days of venoarterial extracorporeal membrane oxygenation (VA-ECMO). VA-ECMO was discontinued 11 days prior to tisagenlecleucel infusion. Shortly thereafter, the patient developed recurrent fever. A CT scan obtained for evaluation of an infectious nidus showed pulmonary opacities with tree-in-bud appearance (possible fungal etiology) and a small intra-abdominal abscess/phlegmon. Given these findings, she was started on posaconazole in addition to existing broad-spectrum antimicrobials (figure 2B). With consideration given to disease burden (11% circulating leukemic blasts on day prior to infusion), optimal antifungal/antimicrobial therapy, and stable clinical exam, the decision was made to proceed with tisagenlecleucel infusion (figure 1B). At the time of infusion, respiratory failure was resolved (without need for non-invasive ventilatory support) and the patient was hemodynamically stable. She remained on amlodipine for management of underlying hypertension, but all inotropic support was discontinued. An echocardiogram (obtained 4 days preinfusion) showed qualitatively normal right ventricular systolic function and mild global hypokinesis of the left ventricle (ejection fraction (EF) 49%, shortening fraction (SF) 32%). The patient’s postinfusion course was complicated by CRS (characterized by fever, cardiac dysfunction, fluid overload, and respiratory insufficiency) and extensive lymphadenopathy (figure 1B) with associated renovascular compromise. She received two doses of tocilizumab on day+21 given 12 hours apart with some clinical improvement, though ultimately with need for resumption of non-invasive ventilatory support and milrinone due to depressed left ventricular systolic function (EF 45%, SF 21.5%). Given the unclear nature of the patient’s aforementioned extensive adenopathy and inability to exclude disease progression, a bone marrow biopsy and aspirate were completed on day 21 postinfusion; it revealed persistent marrow disease, though notably with numerous CD3+ T lymphocytes surrounding visualized blast clusters (figure 4A–E). Follow-up biopsy and aspirate obtained 31 days postinfusion showed extensive fibrosis with no morphological or immunophenotypic evidence of disease (figure 4F). The patient was discharged from the hospital 53 days after tisagenlecleucel infusion. An echocardiogram obtained prior to discharge showed mildly depressed left ventricle systolic function (EF 49%, SF 26%) with mild left ventricular dilation. At the time of discharge (day+54 postinfusion) and on transfer home to her country of origin (day+58), she remained on amlodipine for management of hypertension but was otherwise on no cardiac remodeling medications. She remained on granulocyte colony-stimulating factor for management of severe neutropenia and was transfusion-dependent (packed red blood cells and platelets). In the setting of severe neutropenia, levofloxacin and voriconazole were also continued for infection prophylaxis. Figure 4 Bone marrow biopsy for patient 2 before and after tisagenlecleucel. (A–F) Pathology of biopsies for patient 2. Pathology of the bone marrow at 21 days (A–E) and 31 days (F) after infusion. (A, B) Blasts in the bone marrow exhibited karyorrhexis (blue arrows) mixed with intact peripheral cells 21 days after infusion. (C) CD3+ T cells surrounding and infiltrating leukemic blasts. Blue arrows indicate CD3-negative blast cells. (D) CD19 expression on the blasts. (E) Dual CD3-CD10+ immunostaining highlight T cells in brown (black arrows) and blasts in red (blue arrows). (F) Bone marrow at day 31, depleted of hematopoietic cells. The patient returned to her country of origin 58 days after infusion. Unfortunately, she expired 208 days after infusion due to cardiac arrest of unclear etiology (figure 3B). An autopsy was not obtained. Her last bone marrow evaluation was completed 3 weeks prior to death and showed no morphological or immunophenotypic evidence of disease. Discussion Here, we describe two patients with heavily pretreated r/r B-ALL who were safely infused with tisagenlecleucel despite elevated inflammatory markers, heavy leukemic burden, significant infection, and multiple comorbidities. While our center has experience with infusion of CAR-T cells dating back to 2012 and physician experience with infusions dating to 2005, tisagenlecleucel infusions are and will be the first such infusions for many centers. The complex management of patients prior to, during, and after infusion can be a source of significant concern for many providers, particularly in the presence of underlying infection, evolving disease burden, and/or other confounding variables that increase risk for severe CRS. Early recommendations to sites regarding conditions for infusion and CRS management on the pivotal multisite ELIANA trial of tisagenlecleucel were based on adult and pediatric experience at a single institution. During the trial, experienced providers were frequently consulted by those centers or providers with less experience in CAR-T cell infusion when challenged by cases for which the existing CRS management guidelines did not provide clear guidance on how to proceed (ie, a provider may inquire about tocilizumab for a patient with 5 days of persistent fever to 40°C without other signs or symptoms of CRS). Earlier in the ELIANA trial, there was significant concern that early CRS intervention might lead to diminished efficacy. Through increased patient experience in both trial and real-world settings, however, much has been learned about patient management, including management of mild to severe CRS and neurological toxicity.4 As the number of sites administering tisagenlecleucel expands and a wide range of real-world experience is obtained, it will be difficult to implement rapid changes to official guidelines. In the ELIANA trial, 43% of patients experienced infections within 8 weeks of tisagenlecleucel infusion, and 24% were severe infections (grade 3/4).2 In a few patients, prolonged severe neutropenia led to severe (grade 3 human herpesvirus 6 encephalitis) and fatal (encephalitis and systemic mycosis) infections. Analysis of real-world data in patients with B-ALL from the Center for International Blood and Marrow Transplant Research (CIBMTR) patient registry reported a similar incidence of postinfusion infections (42%), with one fatal bacterial infection.5 Furthermore, management of CRS with short-term tocilizumab therapy (IL-6 blockade) does not appear to increase the risk of infection6; however, it is important to consider that immune responses to invasive fungal infections could involve IL-67 8 and therefore may respond differently to tocilizumab therapy than was reported for our patient. Taken together, these clinical trial and real-world data highlight the known postinfusion infection risk in these patients; however, existing data fail to provide insight into how patients with preinfusion infections might be managed. Expanding the existing literature to further describe management of complex patients who, in some respects, challenge the current guidelines for infusion will be important for improving access to therapy for patients who lack other options for potential durable remission. Technically, the patients reported here were infused with tisagenlecleucel outside of label guidelines due to the presence of elevated inflammatory markers and active infection.1 For patient 1, CRS manifestations were limited to only fever without hypotension or progressive renal or pulmonary involvement. Response of B-ALL to tisagenlecleucel was not compromised by the treatment of CRS at this stage with tocilizumab. For patient 2, CRS was characterized by fever, third-spacing, cardiac dysfunction, and marked lymphadenopathy with secondary renal vascular compromise (suspected to be driven by CAR-T cell expansion). Cardiac dysfunction due to CRS has been observed in children undergoing CAR-T cell therapy, and risk of dysfunction may be increased in patients who have pre-existing cardiac abnormalities; persistent dysfunction remains uncommon, however.9 10 For patient 2, improvement in cardiac function was observed following resolution of CRS, though with unexpected death due to cardiac arrest greater than 200 days after infusion. The etiology of this arrest remains unknown at this time. Through experience gained treating CRS in patients who were part of the ELIANA study, we felt relatively comfortable that we could control CRS and therefore infused both patients under circumstances in which other providers might not have. Nevertheless, tisagenlecleucel infusion was only carried out after extensive counseling regarding the possibility of worst-case scenarios and with serious consideration also given to transport home for palliative measures. In both cases, significant discussions regarding potential life-threatening outcomes were had with patients, families, and medical providers prior to infusion. Conclusions Publication of these case studies and others may guide providers and families as they face similarly challenging circumstances in the future. In addition, they highlight the importance of clear communication with patients, families, and medical team members about potentially lethal adverse events of CAR-T cell therapy when infused in the setting of concurrent life-threatening comorbidities. While these cases represent successful infection management during CAR-T cell therapy, they should not be viewed as typical or even likely outcomes. Treatment of both patients was carried out in a setting with extensive institutional knowledge by an experienced team with the ability to customize standard protocols. Additional reporting of outcomes in patients with active infections via the CIBMTR patient registry is needed to confirm the generalizability of our approach and to better delineate benefit–risk assessment in these patients. Careful reporting on patient experiences and outcomes in the postcommercialization era of tisagenlecleucel will help to ensure safe access to potentially curative therapy for more patients. Ethics statements Patient consent for publication Obtained. Ethics approval This is a retrospective case series for which institutional review board permissions were granted and results were pulled from patients’ charts. Correction notice: This article has been corrected since it first published. The provenance and peer review statement has been included. Contributors: All authors treated patients and contributed to data collection and interpretation. EMH and GDM provided data and helped write the first draft. All authors were involved in revising the manuscript, and read and approved the final version. Funding: Medical writing support was provided by Briana Dye, PhD, of Healthcare Consultancy Group, and was funded by Novartis Pharmaceuticals Corporation. Competing interests: DEY has been an investigator on studies supported by Astellas, Chimerix, Merck, Pfizer, and Viracor-Eurofins. KJA is on the Speaker’s Bureau for and receives support from Novartis Pharmaceuticals. GDM is a consultant and is on the Speaker’s Bureau for Novartis Pharmaceuticals. EMH, RKG, AAA, GSM, SDS, TGF, IAA, WL, and RJH declare that they have no competing interests. Provenance and peer review: Not commissioned; externally peer reviewed. Supplemental material: This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.	8 DOSES	DrugDosageText	CC BY-NC	33472856	19,003,489	2021-01
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug ineffective'.	A 61-Year-Old Woman with Chronic Iron-Deficiency Anemia Due to a Cameron Lesion and a Response to Oral Application of Combined Poloxamer 407 with Hyaluronic Acid and Chondroitin Sulfate Following Single Treatment with Pantoprazole: A Case Report. BACKGROUND Cameron lesions are linear erosions and ulcers on the crests of gastric mucosal folds in the neck of a hiatal hernia and can be difficult to diagnose and treat. This report is of a case of chronic iron deficiency in a 61-year-old woman with a late diagnosis of a Cameron lesion, who did not respond to a single treatment with the proton pump inhibitor (PPI) pantoprazole, but was then treated with oral poloxamer 407 with hyaluronic acid and chondroitin sulfate in addition to PPI. CASE REPORT We report the case of a 61-year-old women with recurrent iron-deficiency anemia, first diagnosed 40 years prior to her presentation at our Endoscopy Unit, and an ongoing melena. We discovered an intrahiatal gastric mucosal defect, which we at first treated with proton pump inhibitors and sucralfate. After a follow-up gastroscopy revealed the persistence of the lesion, we decided to incorporate into the treatment a gel-like substance containing, among others, hyaluronic acid and chondroitin sulfate, and observed that the lesion resolved completely. CONCLUSIONS This report highlights that Cameron lesions should be considered in patients with hiatal hernia who have iron-deficiency anemia and can be diagnosed on upper endoscopy. Further clinical studies are required to determine the role of combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in the management of Cameron lesions. Background In 1986, Cameron and Higgins described linear erosions and ulcers on the crests of gastric mucosal folds in the neck of a hiatal hernia, resulting in gastrointestinal hemorrhage [1]. There is insufficient information on Cameron lesions in the published literature. The scarce data available are from single case reports or case series. Unfortunately, there are no randomized controlled studies on this topic. It is no exaggeration to mention that most textbooks in the field of internal medicine and surgery pay no attention to Cameron lesions, which is a fact that we will probably have to change soon. On the one hand, this is due to the small number of ‘Cameron’ patients worldwide and on the other hand because health care specialists do not recognize them. Even if recognized, a tissue sample from Cameron erosions or ulcers is rarely obtained, which explains the insufficient information about their histopathologic characteristics. We report the case of a 61-year-old woman with chronic iron-deficiency with a late diagnosis of a Cameron lesion, who did not respond to a single treatment with the proton pump inhibitor (PPI) pantoprazole, but was then treated orally with combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in addition to PPI. Case Report A 61-year-old woman presented for an outpatient gastroscopy due to ongoing melena. She took no iron supplementation or drugs that could have changed the stool color. The patient reported no abdominal or defecation problems. Previous surgeries included open appendectomy. The patient had psoriasis vulgaris as a single comorbidity and was a non-smoker. A prophylactic treatment with acetylsalicylic acid (ASA) was applied until 2009 because she had a stroke approximately 2 decades ago (the patient provided no medical records verifying this diagnosis). Two previous gastroscopies (the first at age 21 years and the second approximately 15 years ago) had already been performed because of a chronic ‘intermittent’ iron-deficiency anemia which was first diagnosed about 40 years ago. Both revealed no pathologies. A colonoscopy had also already been performed, with no detectable bleeding sources. After the above-mentioned uneventful endoscopic examinations, the treating physicians attributed the iron deficiency to the excessive menstrual bleeding our patient used to have and prescribed her iron supplementation. Unfortunately, the anemia persisted after menopause. The patient had not taken any iron supplements for 5 years prior to our gastroscopy. Having in mind this medical history, we ordered blood tests. The results are shown in Table 1. During our first gastroscopy, after an unproblematic esophageal intubation, we detected an axial hiatal hernia spreading approximately from 37 to 38 cm from the incisors, with a diameter of 2.2 cm. Advancing aborally, we saw multiple small, flat hematin spots which covered the gastric fundus and corpus, and were not present in the antral area. The duodenum was macroscopically intact. The decisive diagnostic factor proved to be the inversion of the fibroelastic gastroscope, as a small macroscopically visible, not bleeding linear erosion within the hiatal hernia (Figure 1A) was detected. Tissue samples were collected, which revealed only signs of carditis. A therapy with the proton pump inhibitors (PPI) pantoprazole and sucralfate was initiated for 6 weeks (pantoprazole 40 mg b.i.d. for 2 weeks, and afterwards once daily; sucral-fate 4 times daily) and an appointment for a follow-up gastroscopy was made. At the time of the follow-up gastroscopy (6 weeks later), the patient had no complaints and no melena, and all laboratory values were within reference ranges (Table 1). During the endoscopic examination we observed, once more, multiple flat hematin spots in the proximal two-thirds of the stomach. The erosion was still visible, although half of it was covered by fibrin (Figure 1B). We obtained tissue samples from the erosion. We changed the treatment to PPI (pantoprazole 40 mg once daily) and a gel containing, among others, hyaluronic acid, chondroitin sulfate, and poloxamer 407 (4 times daily, 30 min postprandially, and at bedtime) for 3 months. After the second gastroscopy, a new follow-up endoscopic appointment was made, which could not be kept due to the coronavirus crisis. We contacted the patient on the phone. She had no complaints and no melena, and after the above-mentioned 3-month period took only pantoprazole 40 mg once daily. Eventually, a second follow-up gastroscopy was performed 7 months after the initial one. It revealed no erosion, with an intact cardiac mucosa (Figure 1C, 1D). Discussion Our patient had a Cameron erosion that manifested with recurrent anemia and melena. After not achieving a full recovery under the most commonly described treatment with PPI, we decided to incorporate a combination of hyaluronic acid (HA), chondroitin sulfate (CS), and poloxamer 407 (in addition to PPI) into the therapy, and observed that the Cameron erosion disappeared. Cameron lesions are gastric mucosal defects of varying sizes localized within hiatal hernias. They were first described by the American physicians Alan J. Cameron and John A. Higgins (Mayo Clinic, 1986), who found a correlation between diaphragmatic hernias and occult gastrointestinal bleeding. The prevalence of Cameron lesions depends on 2 factors: the size of the hiatal hernia and the size of the hernial sac. Larger hernias are more frequently associated with the development of mucosal defects. Statistically, Cameron lesions are found in 5.2% of all patients with hiatal hernias undergoing gastroscopy, as more than 60% of them present with a single tissue defect. They are diagnosed in up to 50% of all gastroscopic examinations performed for other reasons [2–4]. Etiology and pathophysiology Cameron lesions are a complex phenomenon with an unclear pathogenesis. It is believed that they occur because of the combined effects of extra- and intraluminal mechanical and chemical factors. Pathogenetic factors are briefly presented in Table 2. Morphology Cameron lesions are non-peptic non-GERD-associated mucosal defects occurring on the top of gastric folds over the distal ring of a hiatal hernia (rarely proximally), most often along the lesser gastric curvature. They can be superficial (erosions) or deep (‘riding’ ulcers). Cameron lesions are typically longitudinal, but can also be oblong or ellipsoid, and never comprise the whole circumference of the hiatal hernia [14,15]. Symptoms Most patients with Cameron lesions are older women with chronic anemia and large hiatal hernias. Since hiatal hernias are predominantly asymptomatic, they present with signs of overt (melena, hematochezia, hematemesis) or occult (iron-deficiency anemia, positive fecal occult blood test) gastrointestinal bleeding. Some patients have a medical history of recurrent gastrointestinal bleeding, with or without blood transfusions, oral intake of NSAID, or iron supplements. Rarely, they also have a peptic ulcer or esophagitis [4]. Diagnostics The criterion standard for the diagnosis of Cameron lesions is gastroscopy. Very often, they remain uncovered within an index gastroscopy and are detected during a second-look or repeated gastroscopy (“One only sees what one looks for. One only looks for what one knows.” Goethe). That is why each hiatal hernia should be inspected ante- and retrograde with an additional perpendicular presentation of the hernial opening where the diaphragmatic hiatus exerts the highest pressure on the gastric wall (the predominant localization of Cameron ulcers). The gastroscopist should examine the mucosa above and beneath the Hiatus oesophageus, since Cameron lesions can migrate upwards or downwards depending on the extent to which the stomach is insufflated. The use of chromoendoscopy and optical magnification can also be helpful. Accompanying pathological findings are sometimes observed, such as mucosal edema, erythematous stomach lining, and ecchymoses on gastric folds. There are no specific laboratory tests or imaging modalities for detecting the presence of a Cameron lesion. Blood tests could be normal or demonstrate an iron-deficiency anemia. X-ray or computed tomography may be helpful for the diagnostics of complications (e.g., perforation, volvulus, blood clots in the stomach). Under certain conditions, computed tomography angiogram or Tc-99m-labeled red blood cells scintigraphy can detect an active bleeding source (bleeding speed at least 0.5 ml/min and 0.04 ml/min, respectively). The role of capsule endoscopy remains unclear [12,14]. Histopathologic characteristics To the best of our knowledge, there is only 1 published article containing a description of histopathologic changes in Cameron lesions. Katz et al. describe mucosal alterations due to mucosal vascular obstruction consistent with ischemic gastropathy, such as hemorrhagic infiltrates, fibrin thrombi, inflammatory response, sanguine micro-suffusions, sloughing of epithelial cells, atrophy of crypts, and coagulation necrosis [16]. Our first tissue sampling revealed mucosal alterations consistent with a minor chronic carditis. The results from the second sampling are shown in Figure 2. Management The treatment of Cameron lesions (conservative or surgical) should be determined individually [3]. Because of the limited knowledge about their natural history and the small number of patients, modern medicine was unable to create strict guidelines and algorithms for their management. Literature reports range from cases of spontaneous healing to high-dose PPI treatment regimens [8]. The management of Cameron lesions depends on their clinical presentation. Currently, most authors recommend starting PPI administration as soon as the diagnosis is established. If the patient already receives PPIs once daily, their dosage/frequency of administration should be increased. If blood tests show signs of iron deficiency, iron supplementation is required. In case it has already been initiated, the iron dosage should be increased. A single iron supplementation is not sufficient [17]. By means of the above-mentioned treatment, an adequate symptom control is achieved in up to half of the patients with Cameron lesions. Of course, in case of significant bleeding and hemodynamic shock, stabilizing measures should be taken, since Cameron lesions cause life-threatening gastrointestinal bleeding in up to one-third of all patients [11,18]. NSAID intake should be immediately discontinued. Some authors recommend the administration of prokinetic agents [19]. There are no strict criteria for the conversion from conservative to surgical treatment. Some specialists suggest a secondary surgical treatment in case of persistent complaints (e.g., anemia, hospitalization because of recurrent gastrointestinal bleeding or chronic blood loss) or complications (e.g., refractory bleeding, perforation, volvulus, gastric incarceration) [4]. The main goal of the surgical therapy is to eliminate the mandatory prerequisite for the occurrence of Cameron lesions – the hiatal hernia (there is no Cameron lesion without hiatal hernia.) Possible options are laparoscopic/open fundoplication±gastropexy and the recently developed single-incision transgastric underrunning (SILT) [3,9,20,21]. Endoscopy Endoscopic hemostasis is a relevant but rarely reported non-surgical treatment option for bleeding Cameron lesions. Band ligation and clipping proved to be effective. A follow-up gastroscopy was not performed in many studies, but could be indicated when complaints persist. Our patient After diagnosis establishment, we started a treatment with oral intake of PPIs (pantoprazole 40 mg b.i.d. for 2 weeks and once daily afterwards) in combination with sucralfate 4 times daily, both for 6 weeks. Since the iron panel revealed normal values, no iron supplementation was initiated. PPIs administration aims to inhibit gastric acid production; thus, its possible role in the genesis of Cameron lesions. Sucralfate is a sucrose octasulfate and aluminum hydroxide (AlOH) complex forming a viscous paste in aqueous acidic media (e.g., in the esophagus, stomach, duodenum). It adheres to defective and intact mucosa through polyvalent bridges between negatively charged sucralfate polyanions and positively charged proteins, which are present in high concentrations in mucosal lesions. It also buffers the gastric acid, inhibits the action of pepsin, and absorbs bile salts. All these properties enable sucralfate to act as an effective barrier counteracting the penetration of the above-mentioned substances. Sucralfate proved to be as effective as antacids or H2-receptor antagonists in healing peptic ulcers and in animals, but was ineffective in preventing corticosteroid-induced ulcerations. Sucralfate probably stimulates the local prostaglandin E2 (PGE2) and prostacyclin (PGI2) synthesis in gastric mucosa [22,23]. Since the first follow-up gastroscopy showed no improvement, we decided to use a mixed preparation of hyaluronic acid, chondroitin sulfate, and poloxamer 407. In animal models (Yorkshire pigs), this mixture exhibited positive effects on the healing process of scars left after endoscopic mucosal re-section (EMR) or endoscopic submucosal dissection (ESD) and had been expected to provoke an early proliferation of collagen and elastic fibers in gastric mucosal defects, thus also contributing to ulcer healing. Furthermore, it is an acceptable low-cost alternative to the existing treatment regimens [24]. Hyaluronic acid and chondroitin sulfate are extensively used in wound treatment. Their role in the therapy of gastrointestinal lesions is a subject of continuous research. Hyaluronic acid is a non-sulfated natural glycosaminoglycan and main component of the extracellular matrix. HA is a hygroscopic macromolecule formed by the polymerization of glucuronic acid and N-acetylglucosamine disaccharide. It interacts with several cell surface receptors of malignant and non-malignant gastric mucosa. HA is involved in human innate immunity and inflammatory processes, as it takes part in leukocyte recruitment and macrophage activation. It induces dendritic cell maturation and promotes cytokine release by dendritic cells and endothelial cells. HA also possesses anti-bacterial, anti-fungal, and anti-viral properties and promotes angiogenesis. Topically applied on oral mucosa, it supports tissue hydration in inflammatory processes and mucosal response to tissue injuries that could possibly result in ulcer formation and forms a protective film over exposed mucosal nerve endings, preventing them from overstimulation. Sodium hyaluronate possesses also hemostatic properties when injected in peptic ulcers. Chondroitin sulfate is a glycosaminoglycan consisting of repeated disaccharide units polymerized into long chains. CS is an effective inhibitor of pepsin-induced damage to stomach and duodenal mucosa and showed potent anti-inflammatory properties in animal models [25–27]. Poloxamer 407 is a water-soluble, non-ionic triblock copolymer that is liquid at room temperature and assumes a gel form at body temperature. It is often used as a drug carrier. It possesses mucoadhesive properties and does not irritate mucosal membranes. Poloxamer 407 showed no direct regenerative or antimicrobial properties but maintains a stable concentration of the substances it carries for a prolonged time [28]. Conclusions Hyaluronic acid and chondroitin sulfate are widely used in cosmetics because of their skin regeneration properties. They also possess a proven beneficial effect on the healing of acute and chronic wounds. The role of HA and CS in the treatment of gastrointestinal mucosal pathologies has increasingly been examined over the last decade. In accordance with the published literature and the experience with our patient, we hypothesize that they could also be useful for the conservative treatment of Cameron lesions (in combination with PPI, as stated in the summary of product characteristics of the products approved for use in the gastrointestinal tract). They counteract many of the possible organic and chemical pathogenetic factors, leading to its occurrence, but, of course, cannot influence the most important mechanical factor – the hiatal hernia. In light of the good success of HA and CS in wound healing, we hypothesize the principle that internal wounds (ulcers and mucosal lesions) could be treated like external wounds – treat ulcers like wounds (treat inside like outside). If not achieving a permanent resolution, it could at least gain us some time before surgery to stabilize the patient’s condition, which could sometimes be crucial. This case report highlights that Cameron lesions should be considered in patients with hiatal hernia who have iron-deficiency anemia and can be diagnosed on upper endoscopy. Further clinical studies are required to determine the role of combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in the management of Cameron lesions. The authors would like to express their sincere gratitude to the teams of the Endoscopy Unit, Institute of Pathology, and the Department of General, Visceral and Vascular Surgery of the Steyr Regional Hospital for their help in obtaining and processing all the materials necessary for this article. Conflicts of interest None. Figure 1. (A) Our patient’s Cameron erosion at the time of diagnosis establishment. During the initial gastroscopy, the Cameron erosion presented as a linear mucosal defect (white arrow). After partial gas desufflation the volume and diameter of the sliding hiatal hernia were reduced to provide a better presentation of the lesion and enable sampling. On the left middle and lower part of the image and on the lower right part of the image one can recognize several hematin spots. (B) Our patient’s Cameron erosion during the second (first follow-up) gastroscopy. The left half of the erosion (white arrow) is covered by fibrin, and the right half (black arrow) showed inflammatory alterations (microscopically red mucosa). There are several hematin spots on the middle and lower right side of the image. (C) Image from the last (second follow-up) gastroscopy with maximal gas insufflation. The Cameron erosion is resolved. (D) Image from the last (second follow-up) gastroscopy with maximal gas insufflation. The Cameron erosion is resolved. Figure 2. (A) Microscopic image (HE, magnification ×100) from the second tissue sampling: oxyntocardiac mucosa with minimal foveolar hyperplasia. HE: Hematoxylin and eosin staining. (B) Microscopic image (HE, magnification ×200) from the second tissue sampling: focal minor active inflammatory infiltrates in lamina propria with a slight edema and dilated capillaries. No ischemic stigmata were present. (C) Microscopic image (CAB, magnification x100) from the second tissue sampling: a minimal fibrosis in lamina propria in a single localized area. HP was not detectable. CAB – chromotrope aniline blue staining; HP – Helicobacter pylori. Table 1. Laboratory test results of our patient. The tests were performed at the time of the initial, first, and second follow-up gastroscopies. Unit of measurement Initial gastroscopy First follow-up gastroscopy Second follow-up gastroscopy Normal values WBC G/L 6.95 5.86 4.19 4.00–9.00 Platelets G/L 251 221 270 150–400 RBC T/L 4.43 4.55 4.60 3.80–5.50 Hemoglobin g/dL 14.9 15.0 14.8 12.0–16.5 Hematocrit % 40.3 43.1 41.3 36.0–48.0 MCH pg 33.7 32.9 32.2 27.0–36.0 MCV fL 90.9 94.8 89.9 80.0–101.0 MCHC g/dL 37.1 34.8 35.9 30.0–38.0 RDW-CV % 12.2 12.7 13.9 11.5–15.5 PTT % 96 100 94 70.0–130.0 INR 1.0 1.0 1.0 aPTT sec. 26 25 25 22–35 Fibrinogen g/L 2.99 2.96 3.14 1.80–4.50 TSH mcU/mL 0.91 1.53 0.93 0.36–3.60 CRP mg/L Negative Negative Negative 0–3 K mmol/L 4.6 3.4 3.6 3.5–5.4 Na mmol/L 141 139 139 132–148 Cl mmol/L 107 105 105 95–110 Ca mmol/L 2.3 2.3 2.3 2.0–2.8 Mg mmol/L 1.0 1.0 1.0 0.6–1.1 Creatinine mg/dL 0.7 0.7 0.8 0.5–1.1 eGFR ml/min/l >70 >70 >70 70–150 BUN mg/dL 13 13 12 7–18 Uric acid mg/dL 4.4 4.2 5.2 2.6–6.0 ASAT U/L 21 17 23 0–31 ALAT U/L 20 21 28 0–34 GGT U/L 18 21 22 0–38 AP U/L 125 135 119 50–135 Bilirubin mg/dL 0.9 1.1 1.3 0.2–1.1 Lipase U/L 116 105 106 73–393 Alpha-amylase U/L 53 55 48 25–115 Fe mcg/dL 115 190 119 50–170 Transferrin g/L 2.29 2.41 2.49 2.00–3.60 Transf. sat. % 36 56 34 15–55 Ferritin mcg/L 63 47 57 22–250 Glucose (NF) mg/dl 87 87 97 60–99 WBC – white blood cells; RBC – red blood cells; MCH – mean cell hemoglobin; MCV – mean cell volume; MCHC – mean cell hemoglobin concentration; RDW-CV – red blood cell distribution width; PT – prothrombin time; INR – international normalized ratio; aPTT – activated partial thromboplastin time; TSH – thyroid-stimulating hormone; CRP – C-reactive protein; K – potassium; Na – sodium; Cl – chloride; Ca – calcium; Mg – magnesium; eGFR – estimated glomerular filtration rate; BUN – blood urea nitrogen; ASAT – aspartate aminotransferase; ALAT – alanine aminotransferase; GGT – gamma-glutamyl transferase; AP – alkaline phosphatase; Fe – iron; Transf. sat. – transferrin saturation; NF – non-fasting. Table 2. Pathogeneses of Cameron lesions. Each factor is accompanied by a short explanation of its significance and role in the development of Cameron lesions. Commentary Presence of a diaphragmatic hernia (axial or paraesophageal) This is the most important factor. There are no Cameron lesions without a hiatal hernia. Larger hiatal hernias are associated with a greater risk of their occurrence (10–20% in axial hiatal hernias ≥5 cm) [4] Extraluminal rubbing of the upper stomach and lower esophagus against the diaphragmatic hiatus An important role is played by the permanent extraluminal rubbing of the upper stomach and lower esophagus against the diaphragmatic hiatus (the repeated ascending and descending movement associated with breathing and swallowing) and the intraluminal friction of the mucosal folds within the hiatal hernia against each other and at the level of the distal hernial ring. The gradient between the positive intraabdominal and negative intrathoracic pressure probably provokes an additional sliding movement of the gastro-esophageal junction between both cavities, resulting in mucosal distress with edema, petechiae, and ulcers. This effect is stronger in paraesophageal hiatal hernias Ratio between hernial opening and hernial size From the physical point of view, the ratio between the hernial opening (Hiatus oesophageus) and the hernial size appears to be most important. A significant trauma should be expected in smaller hiatal hernias and a greater friction in larger hernias [5]. The configuration of a hiatal hernia may change with time, which is why erosions or ulcers may appear at different locations [6] Dual-hit hypothesis Multiple studies showed that the mechanical trauma from outside is insufficient to cause mucosal defects. Cameron lesions occur only when the external compression on the gastric wall is combined with an aggressive influence from inside. According to the so-called ‘dual-hit hypothesis’ additional intraluminal and mucosal factors also play a substantial role: gastric acid, gastro-esophageal reflux, and the oral intake of NSAID for 3 days/week within a month. One study demonstrated that in large hiatal hernias, intraluminal lesions occur only when the gastro-esophageal flap valve is intact. This valve counteracts the reflux and affects the role of gastric acid [7,8] Helicobacter pylori There is no clear correlation between HP and Cameron lesions. Only 1/3 of all patients with Cameron lesions are HP-positive [9–11] Gastric ischemia and stasis Additional independent factors for the occurrence of Cameron lesions could be the so-called gastric ‘ischemia’ and ‘stasis’. In some patients, the diaphragmatic hiatus could be narrow enough to cause an intermittent venous stasis in the gastric wall when the diaphragm contracts (e.g., sneezing, coughing, choking). The pathogenesis of Cameron lesions is multifactorial and probably comprises patient’s genotype and phenotype, their comorbidities, and the oral intake of drugs. Patients with Cameron lesions often take iron supplements because the gastric stasis tablets and secretions can remain longer in the herniated stomach and cause pressure erosions [12,13] NSAID – Non-steroidal anti-inflammatory drugs; HP – Helicobacter pylori.	ASPIRIN, PANTOPRAZOLE, SUCRALFATE	DrugsGivenReaction	CC BY-NC-ND	33473099	19,008,761	2021-01-21
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Gastric ulcer'.	A 61-Year-Old Woman with Chronic Iron-Deficiency Anemia Due to a Cameron Lesion and a Response to Oral Application of Combined Poloxamer 407 with Hyaluronic Acid and Chondroitin Sulfate Following Single Treatment with Pantoprazole: A Case Report. BACKGROUND Cameron lesions are linear erosions and ulcers on the crests of gastric mucosal folds in the neck of a hiatal hernia and can be difficult to diagnose and treat. This report is of a case of chronic iron deficiency in a 61-year-old woman with a late diagnosis of a Cameron lesion, who did not respond to a single treatment with the proton pump inhibitor (PPI) pantoprazole, but was then treated with oral poloxamer 407 with hyaluronic acid and chondroitin sulfate in addition to PPI. CASE REPORT We report the case of a 61-year-old women with recurrent iron-deficiency anemia, first diagnosed 40 years prior to her presentation at our Endoscopy Unit, and an ongoing melena. We discovered an intrahiatal gastric mucosal defect, which we at first treated with proton pump inhibitors and sucralfate. After a follow-up gastroscopy revealed the persistence of the lesion, we decided to incorporate into the treatment a gel-like substance containing, among others, hyaluronic acid and chondroitin sulfate, and observed that the lesion resolved completely. CONCLUSIONS This report highlights that Cameron lesions should be considered in patients with hiatal hernia who have iron-deficiency anemia and can be diagnosed on upper endoscopy. Further clinical studies are required to determine the role of combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in the management of Cameron lesions. Background In 1986, Cameron and Higgins described linear erosions and ulcers on the crests of gastric mucosal folds in the neck of a hiatal hernia, resulting in gastrointestinal hemorrhage [1]. There is insufficient information on Cameron lesions in the published literature. The scarce data available are from single case reports or case series. Unfortunately, there are no randomized controlled studies on this topic. It is no exaggeration to mention that most textbooks in the field of internal medicine and surgery pay no attention to Cameron lesions, which is a fact that we will probably have to change soon. On the one hand, this is due to the small number of ‘Cameron’ patients worldwide and on the other hand because health care specialists do not recognize them. Even if recognized, a tissue sample from Cameron erosions or ulcers is rarely obtained, which explains the insufficient information about their histopathologic characteristics. We report the case of a 61-year-old woman with chronic iron-deficiency with a late diagnosis of a Cameron lesion, who did not respond to a single treatment with the proton pump inhibitor (PPI) pantoprazole, but was then treated orally with combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in addition to PPI. Case Report A 61-year-old woman presented for an outpatient gastroscopy due to ongoing melena. She took no iron supplementation or drugs that could have changed the stool color. The patient reported no abdominal or defecation problems. Previous surgeries included open appendectomy. The patient had psoriasis vulgaris as a single comorbidity and was a non-smoker. A prophylactic treatment with acetylsalicylic acid (ASA) was applied until 2009 because she had a stroke approximately 2 decades ago (the patient provided no medical records verifying this diagnosis). Two previous gastroscopies (the first at age 21 years and the second approximately 15 years ago) had already been performed because of a chronic ‘intermittent’ iron-deficiency anemia which was first diagnosed about 40 years ago. Both revealed no pathologies. A colonoscopy had also already been performed, with no detectable bleeding sources. After the above-mentioned uneventful endoscopic examinations, the treating physicians attributed the iron deficiency to the excessive menstrual bleeding our patient used to have and prescribed her iron supplementation. Unfortunately, the anemia persisted after menopause. The patient had not taken any iron supplements for 5 years prior to our gastroscopy. Having in mind this medical history, we ordered blood tests. The results are shown in Table 1. During our first gastroscopy, after an unproblematic esophageal intubation, we detected an axial hiatal hernia spreading approximately from 37 to 38 cm from the incisors, with a diameter of 2.2 cm. Advancing aborally, we saw multiple small, flat hematin spots which covered the gastric fundus and corpus, and were not present in the antral area. The duodenum was macroscopically intact. The decisive diagnostic factor proved to be the inversion of the fibroelastic gastroscope, as a small macroscopically visible, not bleeding linear erosion within the hiatal hernia (Figure 1A) was detected. Tissue samples were collected, which revealed only signs of carditis. A therapy with the proton pump inhibitors (PPI) pantoprazole and sucralfate was initiated for 6 weeks (pantoprazole 40 mg b.i.d. for 2 weeks, and afterwards once daily; sucral-fate 4 times daily) and an appointment for a follow-up gastroscopy was made. At the time of the follow-up gastroscopy (6 weeks later), the patient had no complaints and no melena, and all laboratory values were within reference ranges (Table 1). During the endoscopic examination we observed, once more, multiple flat hematin spots in the proximal two-thirds of the stomach. The erosion was still visible, although half of it was covered by fibrin (Figure 1B). We obtained tissue samples from the erosion. We changed the treatment to PPI (pantoprazole 40 mg once daily) and a gel containing, among others, hyaluronic acid, chondroitin sulfate, and poloxamer 407 (4 times daily, 30 min postprandially, and at bedtime) for 3 months. After the second gastroscopy, a new follow-up endoscopic appointment was made, which could not be kept due to the coronavirus crisis. We contacted the patient on the phone. She had no complaints and no melena, and after the above-mentioned 3-month period took only pantoprazole 40 mg once daily. Eventually, a second follow-up gastroscopy was performed 7 months after the initial one. It revealed no erosion, with an intact cardiac mucosa (Figure 1C, 1D). Discussion Our patient had a Cameron erosion that manifested with recurrent anemia and melena. After not achieving a full recovery under the most commonly described treatment with PPI, we decided to incorporate a combination of hyaluronic acid (HA), chondroitin sulfate (CS), and poloxamer 407 (in addition to PPI) into the therapy, and observed that the Cameron erosion disappeared. Cameron lesions are gastric mucosal defects of varying sizes localized within hiatal hernias. They were first described by the American physicians Alan J. Cameron and John A. Higgins (Mayo Clinic, 1986), who found a correlation between diaphragmatic hernias and occult gastrointestinal bleeding. The prevalence of Cameron lesions depends on 2 factors: the size of the hiatal hernia and the size of the hernial sac. Larger hernias are more frequently associated with the development of mucosal defects. Statistically, Cameron lesions are found in 5.2% of all patients with hiatal hernias undergoing gastroscopy, as more than 60% of them present with a single tissue defect. They are diagnosed in up to 50% of all gastroscopic examinations performed for other reasons [2–4]. Etiology and pathophysiology Cameron lesions are a complex phenomenon with an unclear pathogenesis. It is believed that they occur because of the combined effects of extra- and intraluminal mechanical and chemical factors. Pathogenetic factors are briefly presented in Table 2. Morphology Cameron lesions are non-peptic non-GERD-associated mucosal defects occurring on the top of gastric folds over the distal ring of a hiatal hernia (rarely proximally), most often along the lesser gastric curvature. They can be superficial (erosions) or deep (‘riding’ ulcers). Cameron lesions are typically longitudinal, but can also be oblong or ellipsoid, and never comprise the whole circumference of the hiatal hernia [14,15]. Symptoms Most patients with Cameron lesions are older women with chronic anemia and large hiatal hernias. Since hiatal hernias are predominantly asymptomatic, they present with signs of overt (melena, hematochezia, hematemesis) or occult (iron-deficiency anemia, positive fecal occult blood test) gastrointestinal bleeding. Some patients have a medical history of recurrent gastrointestinal bleeding, with or without blood transfusions, oral intake of NSAID, or iron supplements. Rarely, they also have a peptic ulcer or esophagitis [4]. Diagnostics The criterion standard for the diagnosis of Cameron lesions is gastroscopy. Very often, they remain uncovered within an index gastroscopy and are detected during a second-look or repeated gastroscopy (“One only sees what one looks for. One only looks for what one knows.” Goethe). That is why each hiatal hernia should be inspected ante- and retrograde with an additional perpendicular presentation of the hernial opening where the diaphragmatic hiatus exerts the highest pressure on the gastric wall (the predominant localization of Cameron ulcers). The gastroscopist should examine the mucosa above and beneath the Hiatus oesophageus, since Cameron lesions can migrate upwards or downwards depending on the extent to which the stomach is insufflated. The use of chromoendoscopy and optical magnification can also be helpful. Accompanying pathological findings are sometimes observed, such as mucosal edema, erythematous stomach lining, and ecchymoses on gastric folds. There are no specific laboratory tests or imaging modalities for detecting the presence of a Cameron lesion. Blood tests could be normal or demonstrate an iron-deficiency anemia. X-ray or computed tomography may be helpful for the diagnostics of complications (e.g., perforation, volvulus, blood clots in the stomach). Under certain conditions, computed tomography angiogram or Tc-99m-labeled red blood cells scintigraphy can detect an active bleeding source (bleeding speed at least 0.5 ml/min and 0.04 ml/min, respectively). The role of capsule endoscopy remains unclear [12,14]. Histopathologic characteristics To the best of our knowledge, there is only 1 published article containing a description of histopathologic changes in Cameron lesions. Katz et al. describe mucosal alterations due to mucosal vascular obstruction consistent with ischemic gastropathy, such as hemorrhagic infiltrates, fibrin thrombi, inflammatory response, sanguine micro-suffusions, sloughing of epithelial cells, atrophy of crypts, and coagulation necrosis [16]. Our first tissue sampling revealed mucosal alterations consistent with a minor chronic carditis. The results from the second sampling are shown in Figure 2. Management The treatment of Cameron lesions (conservative or surgical) should be determined individually [3]. Because of the limited knowledge about their natural history and the small number of patients, modern medicine was unable to create strict guidelines and algorithms for their management. Literature reports range from cases of spontaneous healing to high-dose PPI treatment regimens [8]. The management of Cameron lesions depends on their clinical presentation. Currently, most authors recommend starting PPI administration as soon as the diagnosis is established. If the patient already receives PPIs once daily, their dosage/frequency of administration should be increased. If blood tests show signs of iron deficiency, iron supplementation is required. In case it has already been initiated, the iron dosage should be increased. A single iron supplementation is not sufficient [17]. By means of the above-mentioned treatment, an adequate symptom control is achieved in up to half of the patients with Cameron lesions. Of course, in case of significant bleeding and hemodynamic shock, stabilizing measures should be taken, since Cameron lesions cause life-threatening gastrointestinal bleeding in up to one-third of all patients [11,18]. NSAID intake should be immediately discontinued. Some authors recommend the administration of prokinetic agents [19]. There are no strict criteria for the conversion from conservative to surgical treatment. Some specialists suggest a secondary surgical treatment in case of persistent complaints (e.g., anemia, hospitalization because of recurrent gastrointestinal bleeding or chronic blood loss) or complications (e.g., refractory bleeding, perforation, volvulus, gastric incarceration) [4]. The main goal of the surgical therapy is to eliminate the mandatory prerequisite for the occurrence of Cameron lesions – the hiatal hernia (there is no Cameron lesion without hiatal hernia.) Possible options are laparoscopic/open fundoplication±gastropexy and the recently developed single-incision transgastric underrunning (SILT) [3,9,20,21]. Endoscopy Endoscopic hemostasis is a relevant but rarely reported non-surgical treatment option for bleeding Cameron lesions. Band ligation and clipping proved to be effective. A follow-up gastroscopy was not performed in many studies, but could be indicated when complaints persist. Our patient After diagnosis establishment, we started a treatment with oral intake of PPIs (pantoprazole 40 mg b.i.d. for 2 weeks and once daily afterwards) in combination with sucralfate 4 times daily, both for 6 weeks. Since the iron panel revealed normal values, no iron supplementation was initiated. PPIs administration aims to inhibit gastric acid production; thus, its possible role in the genesis of Cameron lesions. Sucralfate is a sucrose octasulfate and aluminum hydroxide (AlOH) complex forming a viscous paste in aqueous acidic media (e.g., in the esophagus, stomach, duodenum). It adheres to defective and intact mucosa through polyvalent bridges between negatively charged sucralfate polyanions and positively charged proteins, which are present in high concentrations in mucosal lesions. It also buffers the gastric acid, inhibits the action of pepsin, and absorbs bile salts. All these properties enable sucralfate to act as an effective barrier counteracting the penetration of the above-mentioned substances. Sucralfate proved to be as effective as antacids or H2-receptor antagonists in healing peptic ulcers and in animals, but was ineffective in preventing corticosteroid-induced ulcerations. Sucralfate probably stimulates the local prostaglandin E2 (PGE2) and prostacyclin (PGI2) synthesis in gastric mucosa [22,23]. Since the first follow-up gastroscopy showed no improvement, we decided to use a mixed preparation of hyaluronic acid, chondroitin sulfate, and poloxamer 407. In animal models (Yorkshire pigs), this mixture exhibited positive effects on the healing process of scars left after endoscopic mucosal re-section (EMR) or endoscopic submucosal dissection (ESD) and had been expected to provoke an early proliferation of collagen and elastic fibers in gastric mucosal defects, thus also contributing to ulcer healing. Furthermore, it is an acceptable low-cost alternative to the existing treatment regimens [24]. Hyaluronic acid and chondroitin sulfate are extensively used in wound treatment. Their role in the therapy of gastrointestinal lesions is a subject of continuous research. Hyaluronic acid is a non-sulfated natural glycosaminoglycan and main component of the extracellular matrix. HA is a hygroscopic macromolecule formed by the polymerization of glucuronic acid and N-acetylglucosamine disaccharide. It interacts with several cell surface receptors of malignant and non-malignant gastric mucosa. HA is involved in human innate immunity and inflammatory processes, as it takes part in leukocyte recruitment and macrophage activation. It induces dendritic cell maturation and promotes cytokine release by dendritic cells and endothelial cells. HA also possesses anti-bacterial, anti-fungal, and anti-viral properties and promotes angiogenesis. Topically applied on oral mucosa, it supports tissue hydration in inflammatory processes and mucosal response to tissue injuries that could possibly result in ulcer formation and forms a protective film over exposed mucosal nerve endings, preventing them from overstimulation. Sodium hyaluronate possesses also hemostatic properties when injected in peptic ulcers. Chondroitin sulfate is a glycosaminoglycan consisting of repeated disaccharide units polymerized into long chains. CS is an effective inhibitor of pepsin-induced damage to stomach and duodenal mucosa and showed potent anti-inflammatory properties in animal models [25–27]. Poloxamer 407 is a water-soluble, non-ionic triblock copolymer that is liquid at room temperature and assumes a gel form at body temperature. It is often used as a drug carrier. It possesses mucoadhesive properties and does not irritate mucosal membranes. Poloxamer 407 showed no direct regenerative or antimicrobial properties but maintains a stable concentration of the substances it carries for a prolonged time [28]. Conclusions Hyaluronic acid and chondroitin sulfate are widely used in cosmetics because of their skin regeneration properties. They also possess a proven beneficial effect on the healing of acute and chronic wounds. The role of HA and CS in the treatment of gastrointestinal mucosal pathologies has increasingly been examined over the last decade. In accordance with the published literature and the experience with our patient, we hypothesize that they could also be useful for the conservative treatment of Cameron lesions (in combination with PPI, as stated in the summary of product characteristics of the products approved for use in the gastrointestinal tract). They counteract many of the possible organic and chemical pathogenetic factors, leading to its occurrence, but, of course, cannot influence the most important mechanical factor – the hiatal hernia. In light of the good success of HA and CS in wound healing, we hypothesize the principle that internal wounds (ulcers and mucosal lesions) could be treated like external wounds – treat ulcers like wounds (treat inside like outside). If not achieving a permanent resolution, it could at least gain us some time before surgery to stabilize the patient’s condition, which could sometimes be crucial. This case report highlights that Cameron lesions should be considered in patients with hiatal hernia who have iron-deficiency anemia and can be diagnosed on upper endoscopy. Further clinical studies are required to determine the role of combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in the management of Cameron lesions. The authors would like to express their sincere gratitude to the teams of the Endoscopy Unit, Institute of Pathology, and the Department of General, Visceral and Vascular Surgery of the Steyr Regional Hospital for their help in obtaining and processing all the materials necessary for this article. Conflicts of interest None. Figure 1. (A) Our patient’s Cameron erosion at the time of diagnosis establishment. During the initial gastroscopy, the Cameron erosion presented as a linear mucosal defect (white arrow). After partial gas desufflation the volume and diameter of the sliding hiatal hernia were reduced to provide a better presentation of the lesion and enable sampling. On the left middle and lower part of the image and on the lower right part of the image one can recognize several hematin spots. (B) Our patient’s Cameron erosion during the second (first follow-up) gastroscopy. The left half of the erosion (white arrow) is covered by fibrin, and the right half (black arrow) showed inflammatory alterations (microscopically red mucosa). There are several hematin spots on the middle and lower right side of the image. (C) Image from the last (second follow-up) gastroscopy with maximal gas insufflation. The Cameron erosion is resolved. (D) Image from the last (second follow-up) gastroscopy with maximal gas insufflation. The Cameron erosion is resolved. Figure 2. (A) Microscopic image (HE, magnification ×100) from the second tissue sampling: oxyntocardiac mucosa with minimal foveolar hyperplasia. HE: Hematoxylin and eosin staining. (B) Microscopic image (HE, magnification ×200) from the second tissue sampling: focal minor active inflammatory infiltrates in lamina propria with a slight edema and dilated capillaries. No ischemic stigmata were present. (C) Microscopic image (CAB, magnification x100) from the second tissue sampling: a minimal fibrosis in lamina propria in a single localized area. HP was not detectable. CAB – chromotrope aniline blue staining; HP – Helicobacter pylori. Table 1. Laboratory test results of our patient. The tests were performed at the time of the initial, first, and second follow-up gastroscopies. Unit of measurement Initial gastroscopy First follow-up gastroscopy Second follow-up gastroscopy Normal values WBC G/L 6.95 5.86 4.19 4.00–9.00 Platelets G/L 251 221 270 150–400 RBC T/L 4.43 4.55 4.60 3.80–5.50 Hemoglobin g/dL 14.9 15.0 14.8 12.0–16.5 Hematocrit % 40.3 43.1 41.3 36.0–48.0 MCH pg 33.7 32.9 32.2 27.0–36.0 MCV fL 90.9 94.8 89.9 80.0–101.0 MCHC g/dL 37.1 34.8 35.9 30.0–38.0 RDW-CV % 12.2 12.7 13.9 11.5–15.5 PTT % 96 100 94 70.0–130.0 INR 1.0 1.0 1.0 aPTT sec. 26 25 25 22–35 Fibrinogen g/L 2.99 2.96 3.14 1.80–4.50 TSH mcU/mL 0.91 1.53 0.93 0.36–3.60 CRP mg/L Negative Negative Negative 0–3 K mmol/L 4.6 3.4 3.6 3.5–5.4 Na mmol/L 141 139 139 132–148 Cl mmol/L 107 105 105 95–110 Ca mmol/L 2.3 2.3 2.3 2.0–2.8 Mg mmol/L 1.0 1.0 1.0 0.6–1.1 Creatinine mg/dL 0.7 0.7 0.8 0.5–1.1 eGFR ml/min/l >70 >70 >70 70–150 BUN mg/dL 13 13 12 7–18 Uric acid mg/dL 4.4 4.2 5.2 2.6–6.0 ASAT U/L 21 17 23 0–31 ALAT U/L 20 21 28 0–34 GGT U/L 18 21 22 0–38 AP U/L 125 135 119 50–135 Bilirubin mg/dL 0.9 1.1 1.3 0.2–1.1 Lipase U/L 116 105 106 73–393 Alpha-amylase U/L 53 55 48 25–115 Fe mcg/dL 115 190 119 50–170 Transferrin g/L 2.29 2.41 2.49 2.00–3.60 Transf. sat. % 36 56 34 15–55 Ferritin mcg/L 63 47 57 22–250 Glucose (NF) mg/dl 87 87 97 60–99 WBC – white blood cells; RBC – red blood cells; MCH – mean cell hemoglobin; MCV – mean cell volume; MCHC – mean cell hemoglobin concentration; RDW-CV – red blood cell distribution width; PT – prothrombin time; INR – international normalized ratio; aPTT – activated partial thromboplastin time; TSH – thyroid-stimulating hormone; CRP – C-reactive protein; K – potassium; Na – sodium; Cl – chloride; Ca – calcium; Mg – magnesium; eGFR – estimated glomerular filtration rate; BUN – blood urea nitrogen; ASAT – aspartate aminotransferase; ALAT – alanine aminotransferase; GGT – gamma-glutamyl transferase; AP – alkaline phosphatase; Fe – iron; Transf. sat. – transferrin saturation; NF – non-fasting. Table 2. Pathogeneses of Cameron lesions. Each factor is accompanied by a short explanation of its significance and role in the development of Cameron lesions. Commentary Presence of a diaphragmatic hernia (axial or paraesophageal) This is the most important factor. There are no Cameron lesions without a hiatal hernia. Larger hiatal hernias are associated with a greater risk of their occurrence (10–20% in axial hiatal hernias ≥5 cm) [4] Extraluminal rubbing of the upper stomach and lower esophagus against the diaphragmatic hiatus An important role is played by the permanent extraluminal rubbing of the upper stomach and lower esophagus against the diaphragmatic hiatus (the repeated ascending and descending movement associated with breathing and swallowing) and the intraluminal friction of the mucosal folds within the hiatal hernia against each other and at the level of the distal hernial ring. The gradient between the positive intraabdominal and negative intrathoracic pressure probably provokes an additional sliding movement of the gastro-esophageal junction between both cavities, resulting in mucosal distress with edema, petechiae, and ulcers. This effect is stronger in paraesophageal hiatal hernias Ratio between hernial opening and hernial size From the physical point of view, the ratio between the hernial opening (Hiatus oesophageus) and the hernial size appears to be most important. A significant trauma should be expected in smaller hiatal hernias and a greater friction in larger hernias [5]. The configuration of a hiatal hernia may change with time, which is why erosions or ulcers may appear at different locations [6] Dual-hit hypothesis Multiple studies showed that the mechanical trauma from outside is insufficient to cause mucosal defects. Cameron lesions occur only when the external compression on the gastric wall is combined with an aggressive influence from inside. According to the so-called ‘dual-hit hypothesis’ additional intraluminal and mucosal factors also play a substantial role: gastric acid, gastro-esophageal reflux, and the oral intake of NSAID for 3 days/week within a month. One study demonstrated that in large hiatal hernias, intraluminal lesions occur only when the gastro-esophageal flap valve is intact. This valve counteracts the reflux and affects the role of gastric acid [7,8] Helicobacter pylori There is no clear correlation between HP and Cameron lesions. Only 1/3 of all patients with Cameron lesions are HP-positive [9–11] Gastric ischemia and stasis Additional independent factors for the occurrence of Cameron lesions could be the so-called gastric ‘ischemia’ and ‘stasis’. In some patients, the diaphragmatic hiatus could be narrow enough to cause an intermittent venous stasis in the gastric wall when the diaphragm contracts (e.g., sneezing, coughing, choking). The pathogenesis of Cameron lesions is multifactorial and probably comprises patient’s genotype and phenotype, their comorbidities, and the oral intake of drugs. Patients with Cameron lesions often take iron supplements because the gastric stasis tablets and secretions can remain longer in the herniated stomach and cause pressure erosions [12,13] NSAID – Non-steroidal anti-inflammatory drugs; HP – Helicobacter pylori.	ASPIRIN, PANTOPRAZOLE, SUCRALFATE	DrugsGivenReaction	CC BY-NC-ND	33473099	19,008,761	2021-01-21
What was the administration route of drug 'PANTOPRAZOLE'?	A 61-Year-Old Woman with Chronic Iron-Deficiency Anemia Due to a Cameron Lesion and a Response to Oral Application of Combined Poloxamer 407 with Hyaluronic Acid and Chondroitin Sulfate Following Single Treatment with Pantoprazole: A Case Report. BACKGROUND Cameron lesions are linear erosions and ulcers on the crests of gastric mucosal folds in the neck of a hiatal hernia and can be difficult to diagnose and treat. This report is of a case of chronic iron deficiency in a 61-year-old woman with a late diagnosis of a Cameron lesion, who did not respond to a single treatment with the proton pump inhibitor (PPI) pantoprazole, but was then treated with oral poloxamer 407 with hyaluronic acid and chondroitin sulfate in addition to PPI. CASE REPORT We report the case of a 61-year-old women with recurrent iron-deficiency anemia, first diagnosed 40 years prior to her presentation at our Endoscopy Unit, and an ongoing melena. We discovered an intrahiatal gastric mucosal defect, which we at first treated with proton pump inhibitors and sucralfate. After a follow-up gastroscopy revealed the persistence of the lesion, we decided to incorporate into the treatment a gel-like substance containing, among others, hyaluronic acid and chondroitin sulfate, and observed that the lesion resolved completely. CONCLUSIONS This report highlights that Cameron lesions should be considered in patients with hiatal hernia who have iron-deficiency anemia and can be diagnosed on upper endoscopy. Further clinical studies are required to determine the role of combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in the management of Cameron lesions. Background In 1986, Cameron and Higgins described linear erosions and ulcers on the crests of gastric mucosal folds in the neck of a hiatal hernia, resulting in gastrointestinal hemorrhage [1]. There is insufficient information on Cameron lesions in the published literature. The scarce data available are from single case reports or case series. Unfortunately, there are no randomized controlled studies on this topic. It is no exaggeration to mention that most textbooks in the field of internal medicine and surgery pay no attention to Cameron lesions, which is a fact that we will probably have to change soon. On the one hand, this is due to the small number of ‘Cameron’ patients worldwide and on the other hand because health care specialists do not recognize them. Even if recognized, a tissue sample from Cameron erosions or ulcers is rarely obtained, which explains the insufficient information about their histopathologic characteristics. We report the case of a 61-year-old woman with chronic iron-deficiency with a late diagnosis of a Cameron lesion, who did not respond to a single treatment with the proton pump inhibitor (PPI) pantoprazole, but was then treated orally with combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in addition to PPI. Case Report A 61-year-old woman presented for an outpatient gastroscopy due to ongoing melena. She took no iron supplementation or drugs that could have changed the stool color. The patient reported no abdominal or defecation problems. Previous surgeries included open appendectomy. The patient had psoriasis vulgaris as a single comorbidity and was a non-smoker. A prophylactic treatment with acetylsalicylic acid (ASA) was applied until 2009 because she had a stroke approximately 2 decades ago (the patient provided no medical records verifying this diagnosis). Two previous gastroscopies (the first at age 21 years and the second approximately 15 years ago) had already been performed because of a chronic ‘intermittent’ iron-deficiency anemia which was first diagnosed about 40 years ago. Both revealed no pathologies. A colonoscopy had also already been performed, with no detectable bleeding sources. After the above-mentioned uneventful endoscopic examinations, the treating physicians attributed the iron deficiency to the excessive menstrual bleeding our patient used to have and prescribed her iron supplementation. Unfortunately, the anemia persisted after menopause. The patient had not taken any iron supplements for 5 years prior to our gastroscopy. Having in mind this medical history, we ordered blood tests. The results are shown in Table 1. During our first gastroscopy, after an unproblematic esophageal intubation, we detected an axial hiatal hernia spreading approximately from 37 to 38 cm from the incisors, with a diameter of 2.2 cm. Advancing aborally, we saw multiple small, flat hematin spots which covered the gastric fundus and corpus, and were not present in the antral area. The duodenum was macroscopically intact. The decisive diagnostic factor proved to be the inversion of the fibroelastic gastroscope, as a small macroscopically visible, not bleeding linear erosion within the hiatal hernia (Figure 1A) was detected. Tissue samples were collected, which revealed only signs of carditis. A therapy with the proton pump inhibitors (PPI) pantoprazole and sucralfate was initiated for 6 weeks (pantoprazole 40 mg b.i.d. for 2 weeks, and afterwards once daily; sucral-fate 4 times daily) and an appointment for a follow-up gastroscopy was made. At the time of the follow-up gastroscopy (6 weeks later), the patient had no complaints and no melena, and all laboratory values were within reference ranges (Table 1). During the endoscopic examination we observed, once more, multiple flat hematin spots in the proximal two-thirds of the stomach. The erosion was still visible, although half of it was covered by fibrin (Figure 1B). We obtained tissue samples from the erosion. We changed the treatment to PPI (pantoprazole 40 mg once daily) and a gel containing, among others, hyaluronic acid, chondroitin sulfate, and poloxamer 407 (4 times daily, 30 min postprandially, and at bedtime) for 3 months. After the second gastroscopy, a new follow-up endoscopic appointment was made, which could not be kept due to the coronavirus crisis. We contacted the patient on the phone. She had no complaints and no melena, and after the above-mentioned 3-month period took only pantoprazole 40 mg once daily. Eventually, a second follow-up gastroscopy was performed 7 months after the initial one. It revealed no erosion, with an intact cardiac mucosa (Figure 1C, 1D). Discussion Our patient had a Cameron erosion that manifested with recurrent anemia and melena. After not achieving a full recovery under the most commonly described treatment with PPI, we decided to incorporate a combination of hyaluronic acid (HA), chondroitin sulfate (CS), and poloxamer 407 (in addition to PPI) into the therapy, and observed that the Cameron erosion disappeared. Cameron lesions are gastric mucosal defects of varying sizes localized within hiatal hernias. They were first described by the American physicians Alan J. Cameron and John A. Higgins (Mayo Clinic, 1986), who found a correlation between diaphragmatic hernias and occult gastrointestinal bleeding. The prevalence of Cameron lesions depends on 2 factors: the size of the hiatal hernia and the size of the hernial sac. Larger hernias are more frequently associated with the development of mucosal defects. Statistically, Cameron lesions are found in 5.2% of all patients with hiatal hernias undergoing gastroscopy, as more than 60% of them present with a single tissue defect. They are diagnosed in up to 50% of all gastroscopic examinations performed for other reasons [2–4]. Etiology and pathophysiology Cameron lesions are a complex phenomenon with an unclear pathogenesis. It is believed that they occur because of the combined effects of extra- and intraluminal mechanical and chemical factors. Pathogenetic factors are briefly presented in Table 2. Morphology Cameron lesions are non-peptic non-GERD-associated mucosal defects occurring on the top of gastric folds over the distal ring of a hiatal hernia (rarely proximally), most often along the lesser gastric curvature. They can be superficial (erosions) or deep (‘riding’ ulcers). Cameron lesions are typically longitudinal, but can also be oblong or ellipsoid, and never comprise the whole circumference of the hiatal hernia [14,15]. Symptoms Most patients with Cameron lesions are older women with chronic anemia and large hiatal hernias. Since hiatal hernias are predominantly asymptomatic, they present with signs of overt (melena, hematochezia, hematemesis) or occult (iron-deficiency anemia, positive fecal occult blood test) gastrointestinal bleeding. Some patients have a medical history of recurrent gastrointestinal bleeding, with or without blood transfusions, oral intake of NSAID, or iron supplements. Rarely, they also have a peptic ulcer or esophagitis [4]. Diagnostics The criterion standard for the diagnosis of Cameron lesions is gastroscopy. Very often, they remain uncovered within an index gastroscopy and are detected during a second-look or repeated gastroscopy (“One only sees what one looks for. One only looks for what one knows.” Goethe). That is why each hiatal hernia should be inspected ante- and retrograde with an additional perpendicular presentation of the hernial opening where the diaphragmatic hiatus exerts the highest pressure on the gastric wall (the predominant localization of Cameron ulcers). The gastroscopist should examine the mucosa above and beneath the Hiatus oesophageus, since Cameron lesions can migrate upwards or downwards depending on the extent to which the stomach is insufflated. The use of chromoendoscopy and optical magnification can also be helpful. Accompanying pathological findings are sometimes observed, such as mucosal edema, erythematous stomach lining, and ecchymoses on gastric folds. There are no specific laboratory tests or imaging modalities for detecting the presence of a Cameron lesion. Blood tests could be normal or demonstrate an iron-deficiency anemia. X-ray or computed tomography may be helpful for the diagnostics of complications (e.g., perforation, volvulus, blood clots in the stomach). Under certain conditions, computed tomography angiogram or Tc-99m-labeled red blood cells scintigraphy can detect an active bleeding source (bleeding speed at least 0.5 ml/min and 0.04 ml/min, respectively). The role of capsule endoscopy remains unclear [12,14]. Histopathologic characteristics To the best of our knowledge, there is only 1 published article containing a description of histopathologic changes in Cameron lesions. Katz et al. describe mucosal alterations due to mucosal vascular obstruction consistent with ischemic gastropathy, such as hemorrhagic infiltrates, fibrin thrombi, inflammatory response, sanguine micro-suffusions, sloughing of epithelial cells, atrophy of crypts, and coagulation necrosis [16]. Our first tissue sampling revealed mucosal alterations consistent with a minor chronic carditis. The results from the second sampling are shown in Figure 2. Management The treatment of Cameron lesions (conservative or surgical) should be determined individually [3]. Because of the limited knowledge about their natural history and the small number of patients, modern medicine was unable to create strict guidelines and algorithms for their management. Literature reports range from cases of spontaneous healing to high-dose PPI treatment regimens [8]. The management of Cameron lesions depends on their clinical presentation. Currently, most authors recommend starting PPI administration as soon as the diagnosis is established. If the patient already receives PPIs once daily, their dosage/frequency of administration should be increased. If blood tests show signs of iron deficiency, iron supplementation is required. In case it has already been initiated, the iron dosage should be increased. A single iron supplementation is not sufficient [17]. By means of the above-mentioned treatment, an adequate symptom control is achieved in up to half of the patients with Cameron lesions. Of course, in case of significant bleeding and hemodynamic shock, stabilizing measures should be taken, since Cameron lesions cause life-threatening gastrointestinal bleeding in up to one-third of all patients [11,18]. NSAID intake should be immediately discontinued. Some authors recommend the administration of prokinetic agents [19]. There are no strict criteria for the conversion from conservative to surgical treatment. Some specialists suggest a secondary surgical treatment in case of persistent complaints (e.g., anemia, hospitalization because of recurrent gastrointestinal bleeding or chronic blood loss) or complications (e.g., refractory bleeding, perforation, volvulus, gastric incarceration) [4]. The main goal of the surgical therapy is to eliminate the mandatory prerequisite for the occurrence of Cameron lesions – the hiatal hernia (there is no Cameron lesion without hiatal hernia.) Possible options are laparoscopic/open fundoplication±gastropexy and the recently developed single-incision transgastric underrunning (SILT) [3,9,20,21]. Endoscopy Endoscopic hemostasis is a relevant but rarely reported non-surgical treatment option for bleeding Cameron lesions. Band ligation and clipping proved to be effective. A follow-up gastroscopy was not performed in many studies, but could be indicated when complaints persist. Our patient After diagnosis establishment, we started a treatment with oral intake of PPIs (pantoprazole 40 mg b.i.d. for 2 weeks and once daily afterwards) in combination with sucralfate 4 times daily, both for 6 weeks. Since the iron panel revealed normal values, no iron supplementation was initiated. PPIs administration aims to inhibit gastric acid production; thus, its possible role in the genesis of Cameron lesions. Sucralfate is a sucrose octasulfate and aluminum hydroxide (AlOH) complex forming a viscous paste in aqueous acidic media (e.g., in the esophagus, stomach, duodenum). It adheres to defective and intact mucosa through polyvalent bridges between negatively charged sucralfate polyanions and positively charged proteins, which are present in high concentrations in mucosal lesions. It also buffers the gastric acid, inhibits the action of pepsin, and absorbs bile salts. All these properties enable sucralfate to act as an effective barrier counteracting the penetration of the above-mentioned substances. Sucralfate proved to be as effective as antacids or H2-receptor antagonists in healing peptic ulcers and in animals, but was ineffective in preventing corticosteroid-induced ulcerations. Sucralfate probably stimulates the local prostaglandin E2 (PGE2) and prostacyclin (PGI2) synthesis in gastric mucosa [22,23]. Since the first follow-up gastroscopy showed no improvement, we decided to use a mixed preparation of hyaluronic acid, chondroitin sulfate, and poloxamer 407. In animal models (Yorkshire pigs), this mixture exhibited positive effects on the healing process of scars left after endoscopic mucosal re-section (EMR) or endoscopic submucosal dissection (ESD) and had been expected to provoke an early proliferation of collagen and elastic fibers in gastric mucosal defects, thus also contributing to ulcer healing. Furthermore, it is an acceptable low-cost alternative to the existing treatment regimens [24]. Hyaluronic acid and chondroitin sulfate are extensively used in wound treatment. Their role in the therapy of gastrointestinal lesions is a subject of continuous research. Hyaluronic acid is a non-sulfated natural glycosaminoglycan and main component of the extracellular matrix. HA is a hygroscopic macromolecule formed by the polymerization of glucuronic acid and N-acetylglucosamine disaccharide. It interacts with several cell surface receptors of malignant and non-malignant gastric mucosa. HA is involved in human innate immunity and inflammatory processes, as it takes part in leukocyte recruitment and macrophage activation. It induces dendritic cell maturation and promotes cytokine release by dendritic cells and endothelial cells. HA also possesses anti-bacterial, anti-fungal, and anti-viral properties and promotes angiogenesis. Topically applied on oral mucosa, it supports tissue hydration in inflammatory processes and mucosal response to tissue injuries that could possibly result in ulcer formation and forms a protective film over exposed mucosal nerve endings, preventing them from overstimulation. Sodium hyaluronate possesses also hemostatic properties when injected in peptic ulcers. Chondroitin sulfate is a glycosaminoglycan consisting of repeated disaccharide units polymerized into long chains. CS is an effective inhibitor of pepsin-induced damage to stomach and duodenal mucosa and showed potent anti-inflammatory properties in animal models [25–27]. Poloxamer 407 is a water-soluble, non-ionic triblock copolymer that is liquid at room temperature and assumes a gel form at body temperature. It is often used as a drug carrier. It possesses mucoadhesive properties and does not irritate mucosal membranes. Poloxamer 407 showed no direct regenerative or antimicrobial properties but maintains a stable concentration of the substances it carries for a prolonged time [28]. Conclusions Hyaluronic acid and chondroitin sulfate are widely used in cosmetics because of their skin regeneration properties. They also possess a proven beneficial effect on the healing of acute and chronic wounds. The role of HA and CS in the treatment of gastrointestinal mucosal pathologies has increasingly been examined over the last decade. In accordance with the published literature and the experience with our patient, we hypothesize that they could also be useful for the conservative treatment of Cameron lesions (in combination with PPI, as stated in the summary of product characteristics of the products approved for use in the gastrointestinal tract). They counteract many of the possible organic and chemical pathogenetic factors, leading to its occurrence, but, of course, cannot influence the most important mechanical factor – the hiatal hernia. In light of the good success of HA and CS in wound healing, we hypothesize the principle that internal wounds (ulcers and mucosal lesions) could be treated like external wounds – treat ulcers like wounds (treat inside like outside). If not achieving a permanent resolution, it could at least gain us some time before surgery to stabilize the patient’s condition, which could sometimes be crucial. This case report highlights that Cameron lesions should be considered in patients with hiatal hernia who have iron-deficiency anemia and can be diagnosed on upper endoscopy. Further clinical studies are required to determine the role of combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in the management of Cameron lesions. The authors would like to express their sincere gratitude to the teams of the Endoscopy Unit, Institute of Pathology, and the Department of General, Visceral and Vascular Surgery of the Steyr Regional Hospital for their help in obtaining and processing all the materials necessary for this article. Conflicts of interest None. Figure 1. (A) Our patient’s Cameron erosion at the time of diagnosis establishment. During the initial gastroscopy, the Cameron erosion presented as a linear mucosal defect (white arrow). After partial gas desufflation the volume and diameter of the sliding hiatal hernia were reduced to provide a better presentation of the lesion and enable sampling. On the left middle and lower part of the image and on the lower right part of the image one can recognize several hematin spots. (B) Our patient’s Cameron erosion during the second (first follow-up) gastroscopy. The left half of the erosion (white arrow) is covered by fibrin, and the right half (black arrow) showed inflammatory alterations (microscopically red mucosa). There are several hematin spots on the middle and lower right side of the image. (C) Image from the last (second follow-up) gastroscopy with maximal gas insufflation. The Cameron erosion is resolved. (D) Image from the last (second follow-up) gastroscopy with maximal gas insufflation. The Cameron erosion is resolved. Figure 2. (A) Microscopic image (HE, magnification ×100) from the second tissue sampling: oxyntocardiac mucosa with minimal foveolar hyperplasia. HE: Hematoxylin and eosin staining. (B) Microscopic image (HE, magnification ×200) from the second tissue sampling: focal minor active inflammatory infiltrates in lamina propria with a slight edema and dilated capillaries. No ischemic stigmata were present. (C) Microscopic image (CAB, magnification x100) from the second tissue sampling: a minimal fibrosis in lamina propria in a single localized area. HP was not detectable. CAB – chromotrope aniline blue staining; HP – Helicobacter pylori. Table 1. Laboratory test results of our patient. The tests were performed at the time of the initial, first, and second follow-up gastroscopies. Unit of measurement Initial gastroscopy First follow-up gastroscopy Second follow-up gastroscopy Normal values WBC G/L 6.95 5.86 4.19 4.00–9.00 Platelets G/L 251 221 270 150–400 RBC T/L 4.43 4.55 4.60 3.80–5.50 Hemoglobin g/dL 14.9 15.0 14.8 12.0–16.5 Hematocrit % 40.3 43.1 41.3 36.0–48.0 MCH pg 33.7 32.9 32.2 27.0–36.0 MCV fL 90.9 94.8 89.9 80.0–101.0 MCHC g/dL 37.1 34.8 35.9 30.0–38.0 RDW-CV % 12.2 12.7 13.9 11.5–15.5 PTT % 96 100 94 70.0–130.0 INR 1.0 1.0 1.0 aPTT sec. 26 25 25 22–35 Fibrinogen g/L 2.99 2.96 3.14 1.80–4.50 TSH mcU/mL 0.91 1.53 0.93 0.36–3.60 CRP mg/L Negative Negative Negative 0–3 K mmol/L 4.6 3.4 3.6 3.5–5.4 Na mmol/L 141 139 139 132–148 Cl mmol/L 107 105 105 95–110 Ca mmol/L 2.3 2.3 2.3 2.0–2.8 Mg mmol/L 1.0 1.0 1.0 0.6–1.1 Creatinine mg/dL 0.7 0.7 0.8 0.5–1.1 eGFR ml/min/l >70 >70 >70 70–150 BUN mg/dL 13 13 12 7–18 Uric acid mg/dL 4.4 4.2 5.2 2.6–6.0 ASAT U/L 21 17 23 0–31 ALAT U/L 20 21 28 0–34 GGT U/L 18 21 22 0–38 AP U/L 125 135 119 50–135 Bilirubin mg/dL 0.9 1.1 1.3 0.2–1.1 Lipase U/L 116 105 106 73–393 Alpha-amylase U/L 53 55 48 25–115 Fe mcg/dL 115 190 119 50–170 Transferrin g/L 2.29 2.41 2.49 2.00–3.60 Transf. sat. % 36 56 34 15–55 Ferritin mcg/L 63 47 57 22–250 Glucose (NF) mg/dl 87 87 97 60–99 WBC – white blood cells; RBC – red blood cells; MCH – mean cell hemoglobin; MCV – mean cell volume; MCHC – mean cell hemoglobin concentration; RDW-CV – red blood cell distribution width; PT – prothrombin time; INR – international normalized ratio; aPTT – activated partial thromboplastin time; TSH – thyroid-stimulating hormone; CRP – C-reactive protein; K – potassium; Na – sodium; Cl – chloride; Ca – calcium; Mg – magnesium; eGFR – estimated glomerular filtration rate; BUN – blood urea nitrogen; ASAT – aspartate aminotransferase; ALAT – alanine aminotransferase; GGT – gamma-glutamyl transferase; AP – alkaline phosphatase; Fe – iron; Transf. sat. – transferrin saturation; NF – non-fasting. Table 2. Pathogeneses of Cameron lesions. Each factor is accompanied by a short explanation of its significance and role in the development of Cameron lesions. Commentary Presence of a diaphragmatic hernia (axial or paraesophageal) This is the most important factor. There are no Cameron lesions without a hiatal hernia. Larger hiatal hernias are associated with a greater risk of their occurrence (10–20% in axial hiatal hernias ≥5 cm) [4] Extraluminal rubbing of the upper stomach and lower esophagus against the diaphragmatic hiatus An important role is played by the permanent extraluminal rubbing of the upper stomach and lower esophagus against the diaphragmatic hiatus (the repeated ascending and descending movement associated with breathing and swallowing) and the intraluminal friction of the mucosal folds within the hiatal hernia against each other and at the level of the distal hernial ring. The gradient between the positive intraabdominal and negative intrathoracic pressure probably provokes an additional sliding movement of the gastro-esophageal junction between both cavities, resulting in mucosal distress with edema, petechiae, and ulcers. This effect is stronger in paraesophageal hiatal hernias Ratio between hernial opening and hernial size From the physical point of view, the ratio between the hernial opening (Hiatus oesophageus) and the hernial size appears to be most important. A significant trauma should be expected in smaller hiatal hernias and a greater friction in larger hernias [5]. The configuration of a hiatal hernia may change with time, which is why erosions or ulcers may appear at different locations [6] Dual-hit hypothesis Multiple studies showed that the mechanical trauma from outside is insufficient to cause mucosal defects. Cameron lesions occur only when the external compression on the gastric wall is combined with an aggressive influence from inside. According to the so-called ‘dual-hit hypothesis’ additional intraluminal and mucosal factors also play a substantial role: gastric acid, gastro-esophageal reflux, and the oral intake of NSAID for 3 days/week within a month. One study demonstrated that in large hiatal hernias, intraluminal lesions occur only when the gastro-esophageal flap valve is intact. This valve counteracts the reflux and affects the role of gastric acid [7,8] Helicobacter pylori There is no clear correlation between HP and Cameron lesions. Only 1/3 of all patients with Cameron lesions are HP-positive [9–11] Gastric ischemia and stasis Additional independent factors for the occurrence of Cameron lesions could be the so-called gastric ‘ischemia’ and ‘stasis’. In some patients, the diaphragmatic hiatus could be narrow enough to cause an intermittent venous stasis in the gastric wall when the diaphragm contracts (e.g., sneezing, coughing, choking). The pathogenesis of Cameron lesions is multifactorial and probably comprises patient’s genotype and phenotype, their comorbidities, and the oral intake of drugs. Patients with Cameron lesions often take iron supplements because the gastric stasis tablets and secretions can remain longer in the herniated stomach and cause pressure erosions [12,13] NSAID – Non-steroidal anti-inflammatory drugs; HP – Helicobacter pylori.	Oral	DrugAdministrationRoute	CC BY-NC-ND	33473099	19,008,761	2021-01-21
What was the dosage of drug 'ASPIRIN'?	A 61-Year-Old Woman with Chronic Iron-Deficiency Anemia Due to a Cameron Lesion and a Response to Oral Application of Combined Poloxamer 407 with Hyaluronic Acid and Chondroitin Sulfate Following Single Treatment with Pantoprazole: A Case Report. BACKGROUND Cameron lesions are linear erosions and ulcers on the crests of gastric mucosal folds in the neck of a hiatal hernia and can be difficult to diagnose and treat. This report is of a case of chronic iron deficiency in a 61-year-old woman with a late diagnosis of a Cameron lesion, who did not respond to a single treatment with the proton pump inhibitor (PPI) pantoprazole, but was then treated with oral poloxamer 407 with hyaluronic acid and chondroitin sulfate in addition to PPI. CASE REPORT We report the case of a 61-year-old women with recurrent iron-deficiency anemia, first diagnosed 40 years prior to her presentation at our Endoscopy Unit, and an ongoing melena. We discovered an intrahiatal gastric mucosal defect, which we at first treated with proton pump inhibitors and sucralfate. After a follow-up gastroscopy revealed the persistence of the lesion, we decided to incorporate into the treatment a gel-like substance containing, among others, hyaluronic acid and chondroitin sulfate, and observed that the lesion resolved completely. CONCLUSIONS This report highlights that Cameron lesions should be considered in patients with hiatal hernia who have iron-deficiency anemia and can be diagnosed on upper endoscopy. Further clinical studies are required to determine the role of combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in the management of Cameron lesions. Background In 1986, Cameron and Higgins described linear erosions and ulcers on the crests of gastric mucosal folds in the neck of a hiatal hernia, resulting in gastrointestinal hemorrhage [1]. There is insufficient information on Cameron lesions in the published literature. The scarce data available are from single case reports or case series. Unfortunately, there are no randomized controlled studies on this topic. It is no exaggeration to mention that most textbooks in the field of internal medicine and surgery pay no attention to Cameron lesions, which is a fact that we will probably have to change soon. On the one hand, this is due to the small number of ‘Cameron’ patients worldwide and on the other hand because health care specialists do not recognize them. Even if recognized, a tissue sample from Cameron erosions or ulcers is rarely obtained, which explains the insufficient information about their histopathologic characteristics. We report the case of a 61-year-old woman with chronic iron-deficiency with a late diagnosis of a Cameron lesion, who did not respond to a single treatment with the proton pump inhibitor (PPI) pantoprazole, but was then treated orally with combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in addition to PPI. Case Report A 61-year-old woman presented for an outpatient gastroscopy due to ongoing melena. She took no iron supplementation or drugs that could have changed the stool color. The patient reported no abdominal or defecation problems. Previous surgeries included open appendectomy. The patient had psoriasis vulgaris as a single comorbidity and was a non-smoker. A prophylactic treatment with acetylsalicylic acid (ASA) was applied until 2009 because she had a stroke approximately 2 decades ago (the patient provided no medical records verifying this diagnosis). Two previous gastroscopies (the first at age 21 years and the second approximately 15 years ago) had already been performed because of a chronic ‘intermittent’ iron-deficiency anemia which was first diagnosed about 40 years ago. Both revealed no pathologies. A colonoscopy had also already been performed, with no detectable bleeding sources. After the above-mentioned uneventful endoscopic examinations, the treating physicians attributed the iron deficiency to the excessive menstrual bleeding our patient used to have and prescribed her iron supplementation. Unfortunately, the anemia persisted after menopause. The patient had not taken any iron supplements for 5 years prior to our gastroscopy. Having in mind this medical history, we ordered blood tests. The results are shown in Table 1. During our first gastroscopy, after an unproblematic esophageal intubation, we detected an axial hiatal hernia spreading approximately from 37 to 38 cm from the incisors, with a diameter of 2.2 cm. Advancing aborally, we saw multiple small, flat hematin spots which covered the gastric fundus and corpus, and were not present in the antral area. The duodenum was macroscopically intact. The decisive diagnostic factor proved to be the inversion of the fibroelastic gastroscope, as a small macroscopically visible, not bleeding linear erosion within the hiatal hernia (Figure 1A) was detected. Tissue samples were collected, which revealed only signs of carditis. A therapy with the proton pump inhibitors (PPI) pantoprazole and sucralfate was initiated for 6 weeks (pantoprazole 40 mg b.i.d. for 2 weeks, and afterwards once daily; sucral-fate 4 times daily) and an appointment for a follow-up gastroscopy was made. At the time of the follow-up gastroscopy (6 weeks later), the patient had no complaints and no melena, and all laboratory values were within reference ranges (Table 1). During the endoscopic examination we observed, once more, multiple flat hematin spots in the proximal two-thirds of the stomach. The erosion was still visible, although half of it was covered by fibrin (Figure 1B). We obtained tissue samples from the erosion. We changed the treatment to PPI (pantoprazole 40 mg once daily) and a gel containing, among others, hyaluronic acid, chondroitin sulfate, and poloxamer 407 (4 times daily, 30 min postprandially, and at bedtime) for 3 months. After the second gastroscopy, a new follow-up endoscopic appointment was made, which could not be kept due to the coronavirus crisis. We contacted the patient on the phone. She had no complaints and no melena, and after the above-mentioned 3-month period took only pantoprazole 40 mg once daily. Eventually, a second follow-up gastroscopy was performed 7 months after the initial one. It revealed no erosion, with an intact cardiac mucosa (Figure 1C, 1D). Discussion Our patient had a Cameron erosion that manifested with recurrent anemia and melena. After not achieving a full recovery under the most commonly described treatment with PPI, we decided to incorporate a combination of hyaluronic acid (HA), chondroitin sulfate (CS), and poloxamer 407 (in addition to PPI) into the therapy, and observed that the Cameron erosion disappeared. Cameron lesions are gastric mucosal defects of varying sizes localized within hiatal hernias. They were first described by the American physicians Alan J. Cameron and John A. Higgins (Mayo Clinic, 1986), who found a correlation between diaphragmatic hernias and occult gastrointestinal bleeding. The prevalence of Cameron lesions depends on 2 factors: the size of the hiatal hernia and the size of the hernial sac. Larger hernias are more frequently associated with the development of mucosal defects. Statistically, Cameron lesions are found in 5.2% of all patients with hiatal hernias undergoing gastroscopy, as more than 60% of them present with a single tissue defect. They are diagnosed in up to 50% of all gastroscopic examinations performed for other reasons [2–4]. Etiology and pathophysiology Cameron lesions are a complex phenomenon with an unclear pathogenesis. It is believed that they occur because of the combined effects of extra- and intraluminal mechanical and chemical factors. Pathogenetic factors are briefly presented in Table 2. Morphology Cameron lesions are non-peptic non-GERD-associated mucosal defects occurring on the top of gastric folds over the distal ring of a hiatal hernia (rarely proximally), most often along the lesser gastric curvature. They can be superficial (erosions) or deep (‘riding’ ulcers). Cameron lesions are typically longitudinal, but can also be oblong or ellipsoid, and never comprise the whole circumference of the hiatal hernia [14,15]. Symptoms Most patients with Cameron lesions are older women with chronic anemia and large hiatal hernias. Since hiatal hernias are predominantly asymptomatic, they present with signs of overt (melena, hematochezia, hematemesis) or occult (iron-deficiency anemia, positive fecal occult blood test) gastrointestinal bleeding. Some patients have a medical history of recurrent gastrointestinal bleeding, with or without blood transfusions, oral intake of NSAID, or iron supplements. Rarely, they also have a peptic ulcer or esophagitis [4]. Diagnostics The criterion standard for the diagnosis of Cameron lesions is gastroscopy. Very often, they remain uncovered within an index gastroscopy and are detected during a second-look or repeated gastroscopy (“One only sees what one looks for. One only looks for what one knows.” Goethe). That is why each hiatal hernia should be inspected ante- and retrograde with an additional perpendicular presentation of the hernial opening where the diaphragmatic hiatus exerts the highest pressure on the gastric wall (the predominant localization of Cameron ulcers). The gastroscopist should examine the mucosa above and beneath the Hiatus oesophageus, since Cameron lesions can migrate upwards or downwards depending on the extent to which the stomach is insufflated. The use of chromoendoscopy and optical magnification can also be helpful. Accompanying pathological findings are sometimes observed, such as mucosal edema, erythematous stomach lining, and ecchymoses on gastric folds. There are no specific laboratory tests or imaging modalities for detecting the presence of a Cameron lesion. Blood tests could be normal or demonstrate an iron-deficiency anemia. X-ray or computed tomography may be helpful for the diagnostics of complications (e.g., perforation, volvulus, blood clots in the stomach). Under certain conditions, computed tomography angiogram or Tc-99m-labeled red blood cells scintigraphy can detect an active bleeding source (bleeding speed at least 0.5 ml/min and 0.04 ml/min, respectively). The role of capsule endoscopy remains unclear [12,14]. Histopathologic characteristics To the best of our knowledge, there is only 1 published article containing a description of histopathologic changes in Cameron lesions. Katz et al. describe mucosal alterations due to mucosal vascular obstruction consistent with ischemic gastropathy, such as hemorrhagic infiltrates, fibrin thrombi, inflammatory response, sanguine micro-suffusions, sloughing of epithelial cells, atrophy of crypts, and coagulation necrosis [16]. Our first tissue sampling revealed mucosal alterations consistent with a minor chronic carditis. The results from the second sampling are shown in Figure 2. Management The treatment of Cameron lesions (conservative or surgical) should be determined individually [3]. Because of the limited knowledge about their natural history and the small number of patients, modern medicine was unable to create strict guidelines and algorithms for their management. Literature reports range from cases of spontaneous healing to high-dose PPI treatment regimens [8]. The management of Cameron lesions depends on their clinical presentation. Currently, most authors recommend starting PPI administration as soon as the diagnosis is established. If the patient already receives PPIs once daily, their dosage/frequency of administration should be increased. If blood tests show signs of iron deficiency, iron supplementation is required. In case it has already been initiated, the iron dosage should be increased. A single iron supplementation is not sufficient [17]. By means of the above-mentioned treatment, an adequate symptom control is achieved in up to half of the patients with Cameron lesions. Of course, in case of significant bleeding and hemodynamic shock, stabilizing measures should be taken, since Cameron lesions cause life-threatening gastrointestinal bleeding in up to one-third of all patients [11,18]. NSAID intake should be immediately discontinued. Some authors recommend the administration of prokinetic agents [19]. There are no strict criteria for the conversion from conservative to surgical treatment. Some specialists suggest a secondary surgical treatment in case of persistent complaints (e.g., anemia, hospitalization because of recurrent gastrointestinal bleeding or chronic blood loss) or complications (e.g., refractory bleeding, perforation, volvulus, gastric incarceration) [4]. The main goal of the surgical therapy is to eliminate the mandatory prerequisite for the occurrence of Cameron lesions – the hiatal hernia (there is no Cameron lesion without hiatal hernia.) Possible options are laparoscopic/open fundoplication±gastropexy and the recently developed single-incision transgastric underrunning (SILT) [3,9,20,21]. Endoscopy Endoscopic hemostasis is a relevant but rarely reported non-surgical treatment option for bleeding Cameron lesions. Band ligation and clipping proved to be effective. A follow-up gastroscopy was not performed in many studies, but could be indicated when complaints persist. Our patient After diagnosis establishment, we started a treatment with oral intake of PPIs (pantoprazole 40 mg b.i.d. for 2 weeks and once daily afterwards) in combination with sucralfate 4 times daily, both for 6 weeks. Since the iron panel revealed normal values, no iron supplementation was initiated. PPIs administration aims to inhibit gastric acid production; thus, its possible role in the genesis of Cameron lesions. Sucralfate is a sucrose octasulfate and aluminum hydroxide (AlOH) complex forming a viscous paste in aqueous acidic media (e.g., in the esophagus, stomach, duodenum). It adheres to defective and intact mucosa through polyvalent bridges between negatively charged sucralfate polyanions and positively charged proteins, which are present in high concentrations in mucosal lesions. It also buffers the gastric acid, inhibits the action of pepsin, and absorbs bile salts. All these properties enable sucralfate to act as an effective barrier counteracting the penetration of the above-mentioned substances. Sucralfate proved to be as effective as antacids or H2-receptor antagonists in healing peptic ulcers and in animals, but was ineffective in preventing corticosteroid-induced ulcerations. Sucralfate probably stimulates the local prostaglandin E2 (PGE2) and prostacyclin (PGI2) synthesis in gastric mucosa [22,23]. Since the first follow-up gastroscopy showed no improvement, we decided to use a mixed preparation of hyaluronic acid, chondroitin sulfate, and poloxamer 407. In animal models (Yorkshire pigs), this mixture exhibited positive effects on the healing process of scars left after endoscopic mucosal re-section (EMR) or endoscopic submucosal dissection (ESD) and had been expected to provoke an early proliferation of collagen and elastic fibers in gastric mucosal defects, thus also contributing to ulcer healing. Furthermore, it is an acceptable low-cost alternative to the existing treatment regimens [24]. Hyaluronic acid and chondroitin sulfate are extensively used in wound treatment. Their role in the therapy of gastrointestinal lesions is a subject of continuous research. Hyaluronic acid is a non-sulfated natural glycosaminoglycan and main component of the extracellular matrix. HA is a hygroscopic macromolecule formed by the polymerization of glucuronic acid and N-acetylglucosamine disaccharide. It interacts with several cell surface receptors of malignant and non-malignant gastric mucosa. HA is involved in human innate immunity and inflammatory processes, as it takes part in leukocyte recruitment and macrophage activation. It induces dendritic cell maturation and promotes cytokine release by dendritic cells and endothelial cells. HA also possesses anti-bacterial, anti-fungal, and anti-viral properties and promotes angiogenesis. Topically applied on oral mucosa, it supports tissue hydration in inflammatory processes and mucosal response to tissue injuries that could possibly result in ulcer formation and forms a protective film over exposed mucosal nerve endings, preventing them from overstimulation. Sodium hyaluronate possesses also hemostatic properties when injected in peptic ulcers. Chondroitin sulfate is a glycosaminoglycan consisting of repeated disaccharide units polymerized into long chains. CS is an effective inhibitor of pepsin-induced damage to stomach and duodenal mucosa and showed potent anti-inflammatory properties in animal models [25–27]. Poloxamer 407 is a water-soluble, non-ionic triblock copolymer that is liquid at room temperature and assumes a gel form at body temperature. It is often used as a drug carrier. It possesses mucoadhesive properties and does not irritate mucosal membranes. Poloxamer 407 showed no direct regenerative or antimicrobial properties but maintains a stable concentration of the substances it carries for a prolonged time [28]. Conclusions Hyaluronic acid and chondroitin sulfate are widely used in cosmetics because of their skin regeneration properties. They also possess a proven beneficial effect on the healing of acute and chronic wounds. The role of HA and CS in the treatment of gastrointestinal mucosal pathologies has increasingly been examined over the last decade. In accordance with the published literature and the experience with our patient, we hypothesize that they could also be useful for the conservative treatment of Cameron lesions (in combination with PPI, as stated in the summary of product characteristics of the products approved for use in the gastrointestinal tract). They counteract many of the possible organic and chemical pathogenetic factors, leading to its occurrence, but, of course, cannot influence the most important mechanical factor – the hiatal hernia. In light of the good success of HA and CS in wound healing, we hypothesize the principle that internal wounds (ulcers and mucosal lesions) could be treated like external wounds – treat ulcers like wounds (treat inside like outside). If not achieving a permanent resolution, it could at least gain us some time before surgery to stabilize the patient’s condition, which could sometimes be crucial. This case report highlights that Cameron lesions should be considered in patients with hiatal hernia who have iron-deficiency anemia and can be diagnosed on upper endoscopy. Further clinical studies are required to determine the role of combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in the management of Cameron lesions. The authors would like to express their sincere gratitude to the teams of the Endoscopy Unit, Institute of Pathology, and the Department of General, Visceral and Vascular Surgery of the Steyr Regional Hospital for their help in obtaining and processing all the materials necessary for this article. Conflicts of interest None. Figure 1. (A) Our patient’s Cameron erosion at the time of diagnosis establishment. During the initial gastroscopy, the Cameron erosion presented as a linear mucosal defect (white arrow). After partial gas desufflation the volume and diameter of the sliding hiatal hernia were reduced to provide a better presentation of the lesion and enable sampling. On the left middle and lower part of the image and on the lower right part of the image one can recognize several hematin spots. (B) Our patient’s Cameron erosion during the second (first follow-up) gastroscopy. The left half of the erosion (white arrow) is covered by fibrin, and the right half (black arrow) showed inflammatory alterations (microscopically red mucosa). There are several hematin spots on the middle and lower right side of the image. (C) Image from the last (second follow-up) gastroscopy with maximal gas insufflation. The Cameron erosion is resolved. (D) Image from the last (second follow-up) gastroscopy with maximal gas insufflation. The Cameron erosion is resolved. Figure 2. (A) Microscopic image (HE, magnification ×100) from the second tissue sampling: oxyntocardiac mucosa with minimal foveolar hyperplasia. HE: Hematoxylin and eosin staining. (B) Microscopic image (HE, magnification ×200) from the second tissue sampling: focal minor active inflammatory infiltrates in lamina propria with a slight edema and dilated capillaries. No ischemic stigmata were present. (C) Microscopic image (CAB, magnification x100) from the second tissue sampling: a minimal fibrosis in lamina propria in a single localized area. HP was not detectable. CAB – chromotrope aniline blue staining; HP – Helicobacter pylori. Table 1. Laboratory test results of our patient. The tests were performed at the time of the initial, first, and second follow-up gastroscopies. Unit of measurement Initial gastroscopy First follow-up gastroscopy Second follow-up gastroscopy Normal values WBC G/L 6.95 5.86 4.19 4.00–9.00 Platelets G/L 251 221 270 150–400 RBC T/L 4.43 4.55 4.60 3.80–5.50 Hemoglobin g/dL 14.9 15.0 14.8 12.0–16.5 Hematocrit % 40.3 43.1 41.3 36.0–48.0 MCH pg 33.7 32.9 32.2 27.0–36.0 MCV fL 90.9 94.8 89.9 80.0–101.0 MCHC g/dL 37.1 34.8 35.9 30.0–38.0 RDW-CV % 12.2 12.7 13.9 11.5–15.5 PTT % 96 100 94 70.0–130.0 INR 1.0 1.0 1.0 aPTT sec. 26 25 25 22–35 Fibrinogen g/L 2.99 2.96 3.14 1.80–4.50 TSH mcU/mL 0.91 1.53 0.93 0.36–3.60 CRP mg/L Negative Negative Negative 0–3 K mmol/L 4.6 3.4 3.6 3.5–5.4 Na mmol/L 141 139 139 132–148 Cl mmol/L 107 105 105 95–110 Ca mmol/L 2.3 2.3 2.3 2.0–2.8 Mg mmol/L 1.0 1.0 1.0 0.6–1.1 Creatinine mg/dL 0.7 0.7 0.8 0.5–1.1 eGFR ml/min/l >70 >70 >70 70–150 BUN mg/dL 13 13 12 7–18 Uric acid mg/dL 4.4 4.2 5.2 2.6–6.0 ASAT U/L 21 17 23 0–31 ALAT U/L 20 21 28 0–34 GGT U/L 18 21 22 0–38 AP U/L 125 135 119 50–135 Bilirubin mg/dL 0.9 1.1 1.3 0.2–1.1 Lipase U/L 116 105 106 73–393 Alpha-amylase U/L 53 55 48 25–115 Fe mcg/dL 115 190 119 50–170 Transferrin g/L 2.29 2.41 2.49 2.00–3.60 Transf. sat. % 36 56 34 15–55 Ferritin mcg/L 63 47 57 22–250 Glucose (NF) mg/dl 87 87 97 60–99 WBC – white blood cells; RBC – red blood cells; MCH – mean cell hemoglobin; MCV – mean cell volume; MCHC – mean cell hemoglobin concentration; RDW-CV – red blood cell distribution width; PT – prothrombin time; INR – international normalized ratio; aPTT – activated partial thromboplastin time; TSH – thyroid-stimulating hormone; CRP – C-reactive protein; K – potassium; Na – sodium; Cl – chloride; Ca – calcium; Mg – magnesium; eGFR – estimated glomerular filtration rate; BUN – blood urea nitrogen; ASAT – aspartate aminotransferase; ALAT – alanine aminotransferase; GGT – gamma-glutamyl transferase; AP – alkaline phosphatase; Fe – iron; Transf. sat. – transferrin saturation; NF – non-fasting. Table 2. Pathogeneses of Cameron lesions. Each factor is accompanied by a short explanation of its significance and role in the development of Cameron lesions. Commentary Presence of a diaphragmatic hernia (axial or paraesophageal) This is the most important factor. There are no Cameron lesions without a hiatal hernia. Larger hiatal hernias are associated with a greater risk of their occurrence (10–20% in axial hiatal hernias ≥5 cm) [4] Extraluminal rubbing of the upper stomach and lower esophagus against the diaphragmatic hiatus An important role is played by the permanent extraluminal rubbing of the upper stomach and lower esophagus against the diaphragmatic hiatus (the repeated ascending and descending movement associated with breathing and swallowing) and the intraluminal friction of the mucosal folds within the hiatal hernia against each other and at the level of the distal hernial ring. The gradient between the positive intraabdominal and negative intrathoracic pressure probably provokes an additional sliding movement of the gastro-esophageal junction between both cavities, resulting in mucosal distress with edema, petechiae, and ulcers. This effect is stronger in paraesophageal hiatal hernias Ratio between hernial opening and hernial size From the physical point of view, the ratio between the hernial opening (Hiatus oesophageus) and the hernial size appears to be most important. A significant trauma should be expected in smaller hiatal hernias and a greater friction in larger hernias [5]. The configuration of a hiatal hernia may change with time, which is why erosions or ulcers may appear at different locations [6] Dual-hit hypothesis Multiple studies showed that the mechanical trauma from outside is insufficient to cause mucosal defects. Cameron lesions occur only when the external compression on the gastric wall is combined with an aggressive influence from inside. According to the so-called ‘dual-hit hypothesis’ additional intraluminal and mucosal factors also play a substantial role: gastric acid, gastro-esophageal reflux, and the oral intake of NSAID for 3 days/week within a month. One study demonstrated that in large hiatal hernias, intraluminal lesions occur only when the gastro-esophageal flap valve is intact. This valve counteracts the reflux and affects the role of gastric acid [7,8] Helicobacter pylori There is no clear correlation between HP and Cameron lesions. Only 1/3 of all patients with Cameron lesions are HP-positive [9–11] Gastric ischemia and stasis Additional independent factors for the occurrence of Cameron lesions could be the so-called gastric ‘ischemia’ and ‘stasis’. In some patients, the diaphragmatic hiatus could be narrow enough to cause an intermittent venous stasis in the gastric wall when the diaphragm contracts (e.g., sneezing, coughing, choking). The pathogenesis of Cameron lesions is multifactorial and probably comprises patient’s genotype and phenotype, their comorbidities, and the oral intake of drugs. Patients with Cameron lesions often take iron supplements because the gastric stasis tablets and secretions can remain longer in the herniated stomach and cause pressure erosions [12,13] NSAID – Non-steroidal anti-inflammatory drugs; HP – Helicobacter pylori.	UNTIL 2009	DrugDosageText	CC BY-NC-ND	33473099	19,008,761	2021-01-21
What was the dosage of drug 'SUCRALFATE'?	A 61-Year-Old Woman with Chronic Iron-Deficiency Anemia Due to a Cameron Lesion and a Response to Oral Application of Combined Poloxamer 407 with Hyaluronic Acid and Chondroitin Sulfate Following Single Treatment with Pantoprazole: A Case Report. BACKGROUND Cameron lesions are linear erosions and ulcers on the crests of gastric mucosal folds in the neck of a hiatal hernia and can be difficult to diagnose and treat. This report is of a case of chronic iron deficiency in a 61-year-old woman with a late diagnosis of a Cameron lesion, who did not respond to a single treatment with the proton pump inhibitor (PPI) pantoprazole, but was then treated with oral poloxamer 407 with hyaluronic acid and chondroitin sulfate in addition to PPI. CASE REPORT We report the case of a 61-year-old women with recurrent iron-deficiency anemia, first diagnosed 40 years prior to her presentation at our Endoscopy Unit, and an ongoing melena. We discovered an intrahiatal gastric mucosal defect, which we at first treated with proton pump inhibitors and sucralfate. After a follow-up gastroscopy revealed the persistence of the lesion, we decided to incorporate into the treatment a gel-like substance containing, among others, hyaluronic acid and chondroitin sulfate, and observed that the lesion resolved completely. CONCLUSIONS This report highlights that Cameron lesions should be considered in patients with hiatal hernia who have iron-deficiency anemia and can be diagnosed on upper endoscopy. Further clinical studies are required to determine the role of combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in the management of Cameron lesions. Background In 1986, Cameron and Higgins described linear erosions and ulcers on the crests of gastric mucosal folds in the neck of a hiatal hernia, resulting in gastrointestinal hemorrhage [1]. There is insufficient information on Cameron lesions in the published literature. The scarce data available are from single case reports or case series. Unfortunately, there are no randomized controlled studies on this topic. It is no exaggeration to mention that most textbooks in the field of internal medicine and surgery pay no attention to Cameron lesions, which is a fact that we will probably have to change soon. On the one hand, this is due to the small number of ‘Cameron’ patients worldwide and on the other hand because health care specialists do not recognize them. Even if recognized, a tissue sample from Cameron erosions or ulcers is rarely obtained, which explains the insufficient information about their histopathologic characteristics. We report the case of a 61-year-old woman with chronic iron-deficiency with a late diagnosis of a Cameron lesion, who did not respond to a single treatment with the proton pump inhibitor (PPI) pantoprazole, but was then treated orally with combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in addition to PPI. Case Report A 61-year-old woman presented for an outpatient gastroscopy due to ongoing melena. She took no iron supplementation or drugs that could have changed the stool color. The patient reported no abdominal or defecation problems. Previous surgeries included open appendectomy. The patient had psoriasis vulgaris as a single comorbidity and was a non-smoker. A prophylactic treatment with acetylsalicylic acid (ASA) was applied until 2009 because she had a stroke approximately 2 decades ago (the patient provided no medical records verifying this diagnosis). Two previous gastroscopies (the first at age 21 years and the second approximately 15 years ago) had already been performed because of a chronic ‘intermittent’ iron-deficiency anemia which was first diagnosed about 40 years ago. Both revealed no pathologies. A colonoscopy had also already been performed, with no detectable bleeding sources. After the above-mentioned uneventful endoscopic examinations, the treating physicians attributed the iron deficiency to the excessive menstrual bleeding our patient used to have and prescribed her iron supplementation. Unfortunately, the anemia persisted after menopause. The patient had not taken any iron supplements for 5 years prior to our gastroscopy. Having in mind this medical history, we ordered blood tests. The results are shown in Table 1. During our first gastroscopy, after an unproblematic esophageal intubation, we detected an axial hiatal hernia spreading approximately from 37 to 38 cm from the incisors, with a diameter of 2.2 cm. Advancing aborally, we saw multiple small, flat hematin spots which covered the gastric fundus and corpus, and were not present in the antral area. The duodenum was macroscopically intact. The decisive diagnostic factor proved to be the inversion of the fibroelastic gastroscope, as a small macroscopically visible, not bleeding linear erosion within the hiatal hernia (Figure 1A) was detected. Tissue samples were collected, which revealed only signs of carditis. A therapy with the proton pump inhibitors (PPI) pantoprazole and sucralfate was initiated for 6 weeks (pantoprazole 40 mg b.i.d. for 2 weeks, and afterwards once daily; sucral-fate 4 times daily) and an appointment for a follow-up gastroscopy was made. At the time of the follow-up gastroscopy (6 weeks later), the patient had no complaints and no melena, and all laboratory values were within reference ranges (Table 1). During the endoscopic examination we observed, once more, multiple flat hematin spots in the proximal two-thirds of the stomach. The erosion was still visible, although half of it was covered by fibrin (Figure 1B). We obtained tissue samples from the erosion. We changed the treatment to PPI (pantoprazole 40 mg once daily) and a gel containing, among others, hyaluronic acid, chondroitin sulfate, and poloxamer 407 (4 times daily, 30 min postprandially, and at bedtime) for 3 months. After the second gastroscopy, a new follow-up endoscopic appointment was made, which could not be kept due to the coronavirus crisis. We contacted the patient on the phone. She had no complaints and no melena, and after the above-mentioned 3-month period took only pantoprazole 40 mg once daily. Eventually, a second follow-up gastroscopy was performed 7 months after the initial one. It revealed no erosion, with an intact cardiac mucosa (Figure 1C, 1D). Discussion Our patient had a Cameron erosion that manifested with recurrent anemia and melena. After not achieving a full recovery under the most commonly described treatment with PPI, we decided to incorporate a combination of hyaluronic acid (HA), chondroitin sulfate (CS), and poloxamer 407 (in addition to PPI) into the therapy, and observed that the Cameron erosion disappeared. Cameron lesions are gastric mucosal defects of varying sizes localized within hiatal hernias. They were first described by the American physicians Alan J. Cameron and John A. Higgins (Mayo Clinic, 1986), who found a correlation between diaphragmatic hernias and occult gastrointestinal bleeding. The prevalence of Cameron lesions depends on 2 factors: the size of the hiatal hernia and the size of the hernial sac. Larger hernias are more frequently associated with the development of mucosal defects. Statistically, Cameron lesions are found in 5.2% of all patients with hiatal hernias undergoing gastroscopy, as more than 60% of them present with a single tissue defect. They are diagnosed in up to 50% of all gastroscopic examinations performed for other reasons [2–4]. Etiology and pathophysiology Cameron lesions are a complex phenomenon with an unclear pathogenesis. It is believed that they occur because of the combined effects of extra- and intraluminal mechanical and chemical factors. Pathogenetic factors are briefly presented in Table 2. Morphology Cameron lesions are non-peptic non-GERD-associated mucosal defects occurring on the top of gastric folds over the distal ring of a hiatal hernia (rarely proximally), most often along the lesser gastric curvature. They can be superficial (erosions) or deep (‘riding’ ulcers). Cameron lesions are typically longitudinal, but can also be oblong or ellipsoid, and never comprise the whole circumference of the hiatal hernia [14,15]. Symptoms Most patients with Cameron lesions are older women with chronic anemia and large hiatal hernias. Since hiatal hernias are predominantly asymptomatic, they present with signs of overt (melena, hematochezia, hematemesis) or occult (iron-deficiency anemia, positive fecal occult blood test) gastrointestinal bleeding. Some patients have a medical history of recurrent gastrointestinal bleeding, with or without blood transfusions, oral intake of NSAID, or iron supplements. Rarely, they also have a peptic ulcer or esophagitis [4]. Diagnostics The criterion standard for the diagnosis of Cameron lesions is gastroscopy. Very often, they remain uncovered within an index gastroscopy and are detected during a second-look or repeated gastroscopy (“One only sees what one looks for. One only looks for what one knows.” Goethe). That is why each hiatal hernia should be inspected ante- and retrograde with an additional perpendicular presentation of the hernial opening where the diaphragmatic hiatus exerts the highest pressure on the gastric wall (the predominant localization of Cameron ulcers). The gastroscopist should examine the mucosa above and beneath the Hiatus oesophageus, since Cameron lesions can migrate upwards or downwards depending on the extent to which the stomach is insufflated. The use of chromoendoscopy and optical magnification can also be helpful. Accompanying pathological findings are sometimes observed, such as mucosal edema, erythematous stomach lining, and ecchymoses on gastric folds. There are no specific laboratory tests or imaging modalities for detecting the presence of a Cameron lesion. Blood tests could be normal or demonstrate an iron-deficiency anemia. X-ray or computed tomography may be helpful for the diagnostics of complications (e.g., perforation, volvulus, blood clots in the stomach). Under certain conditions, computed tomography angiogram or Tc-99m-labeled red blood cells scintigraphy can detect an active bleeding source (bleeding speed at least 0.5 ml/min and 0.04 ml/min, respectively). The role of capsule endoscopy remains unclear [12,14]. Histopathologic characteristics To the best of our knowledge, there is only 1 published article containing a description of histopathologic changes in Cameron lesions. Katz et al. describe mucosal alterations due to mucosal vascular obstruction consistent with ischemic gastropathy, such as hemorrhagic infiltrates, fibrin thrombi, inflammatory response, sanguine micro-suffusions, sloughing of epithelial cells, atrophy of crypts, and coagulation necrosis [16]. Our first tissue sampling revealed mucosal alterations consistent with a minor chronic carditis. The results from the second sampling are shown in Figure 2. Management The treatment of Cameron lesions (conservative or surgical) should be determined individually [3]. Because of the limited knowledge about their natural history and the small number of patients, modern medicine was unable to create strict guidelines and algorithms for their management. Literature reports range from cases of spontaneous healing to high-dose PPI treatment regimens [8]. The management of Cameron lesions depends on their clinical presentation. Currently, most authors recommend starting PPI administration as soon as the diagnosis is established. If the patient already receives PPIs once daily, their dosage/frequency of administration should be increased. If blood tests show signs of iron deficiency, iron supplementation is required. In case it has already been initiated, the iron dosage should be increased. A single iron supplementation is not sufficient [17]. By means of the above-mentioned treatment, an adequate symptom control is achieved in up to half of the patients with Cameron lesions. Of course, in case of significant bleeding and hemodynamic shock, stabilizing measures should be taken, since Cameron lesions cause life-threatening gastrointestinal bleeding in up to one-third of all patients [11,18]. NSAID intake should be immediately discontinued. Some authors recommend the administration of prokinetic agents [19]. There are no strict criteria for the conversion from conservative to surgical treatment. Some specialists suggest a secondary surgical treatment in case of persistent complaints (e.g., anemia, hospitalization because of recurrent gastrointestinal bleeding or chronic blood loss) or complications (e.g., refractory bleeding, perforation, volvulus, gastric incarceration) [4]. The main goal of the surgical therapy is to eliminate the mandatory prerequisite for the occurrence of Cameron lesions – the hiatal hernia (there is no Cameron lesion without hiatal hernia.) Possible options are laparoscopic/open fundoplication±gastropexy and the recently developed single-incision transgastric underrunning (SILT) [3,9,20,21]. Endoscopy Endoscopic hemostasis is a relevant but rarely reported non-surgical treatment option for bleeding Cameron lesions. Band ligation and clipping proved to be effective. A follow-up gastroscopy was not performed in many studies, but could be indicated when complaints persist. Our patient After diagnosis establishment, we started a treatment with oral intake of PPIs (pantoprazole 40 mg b.i.d. for 2 weeks and once daily afterwards) in combination with sucralfate 4 times daily, both for 6 weeks. Since the iron panel revealed normal values, no iron supplementation was initiated. PPIs administration aims to inhibit gastric acid production; thus, its possible role in the genesis of Cameron lesions. Sucralfate is a sucrose octasulfate and aluminum hydroxide (AlOH) complex forming a viscous paste in aqueous acidic media (e.g., in the esophagus, stomach, duodenum). It adheres to defective and intact mucosa through polyvalent bridges between negatively charged sucralfate polyanions and positively charged proteins, which are present in high concentrations in mucosal lesions. It also buffers the gastric acid, inhibits the action of pepsin, and absorbs bile salts. All these properties enable sucralfate to act as an effective barrier counteracting the penetration of the above-mentioned substances. Sucralfate proved to be as effective as antacids or H2-receptor antagonists in healing peptic ulcers and in animals, but was ineffective in preventing corticosteroid-induced ulcerations. Sucralfate probably stimulates the local prostaglandin E2 (PGE2) and prostacyclin (PGI2) synthesis in gastric mucosa [22,23]. Since the first follow-up gastroscopy showed no improvement, we decided to use a mixed preparation of hyaluronic acid, chondroitin sulfate, and poloxamer 407. In animal models (Yorkshire pigs), this mixture exhibited positive effects on the healing process of scars left after endoscopic mucosal re-section (EMR) or endoscopic submucosal dissection (ESD) and had been expected to provoke an early proliferation of collagen and elastic fibers in gastric mucosal defects, thus also contributing to ulcer healing. Furthermore, it is an acceptable low-cost alternative to the existing treatment regimens [24]. Hyaluronic acid and chondroitin sulfate are extensively used in wound treatment. Their role in the therapy of gastrointestinal lesions is a subject of continuous research. Hyaluronic acid is a non-sulfated natural glycosaminoglycan and main component of the extracellular matrix. HA is a hygroscopic macromolecule formed by the polymerization of glucuronic acid and N-acetylglucosamine disaccharide. It interacts with several cell surface receptors of malignant and non-malignant gastric mucosa. HA is involved in human innate immunity and inflammatory processes, as it takes part in leukocyte recruitment and macrophage activation. It induces dendritic cell maturation and promotes cytokine release by dendritic cells and endothelial cells. HA also possesses anti-bacterial, anti-fungal, and anti-viral properties and promotes angiogenesis. Topically applied on oral mucosa, it supports tissue hydration in inflammatory processes and mucosal response to tissue injuries that could possibly result in ulcer formation and forms a protective film over exposed mucosal nerve endings, preventing them from overstimulation. Sodium hyaluronate possesses also hemostatic properties when injected in peptic ulcers. Chondroitin sulfate is a glycosaminoglycan consisting of repeated disaccharide units polymerized into long chains. CS is an effective inhibitor of pepsin-induced damage to stomach and duodenal mucosa and showed potent anti-inflammatory properties in animal models [25–27]. Poloxamer 407 is a water-soluble, non-ionic triblock copolymer that is liquid at room temperature and assumes a gel form at body temperature. It is often used as a drug carrier. It possesses mucoadhesive properties and does not irritate mucosal membranes. Poloxamer 407 showed no direct regenerative or antimicrobial properties but maintains a stable concentration of the substances it carries for a prolonged time [28]. Conclusions Hyaluronic acid and chondroitin sulfate are widely used in cosmetics because of their skin regeneration properties. They also possess a proven beneficial effect on the healing of acute and chronic wounds. The role of HA and CS in the treatment of gastrointestinal mucosal pathologies has increasingly been examined over the last decade. In accordance with the published literature and the experience with our patient, we hypothesize that they could also be useful for the conservative treatment of Cameron lesions (in combination with PPI, as stated in the summary of product characteristics of the products approved for use in the gastrointestinal tract). They counteract many of the possible organic and chemical pathogenetic factors, leading to its occurrence, but, of course, cannot influence the most important mechanical factor – the hiatal hernia. In light of the good success of HA and CS in wound healing, we hypothesize the principle that internal wounds (ulcers and mucosal lesions) could be treated like external wounds – treat ulcers like wounds (treat inside like outside). If not achieving a permanent resolution, it could at least gain us some time before surgery to stabilize the patient’s condition, which could sometimes be crucial. This case report highlights that Cameron lesions should be considered in patients with hiatal hernia who have iron-deficiency anemia and can be diagnosed on upper endoscopy. Further clinical studies are required to determine the role of combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in the management of Cameron lesions. The authors would like to express their sincere gratitude to the teams of the Endoscopy Unit, Institute of Pathology, and the Department of General, Visceral and Vascular Surgery of the Steyr Regional Hospital for their help in obtaining and processing all the materials necessary for this article. Conflicts of interest None. Figure 1. (A) Our patient’s Cameron erosion at the time of diagnosis establishment. During the initial gastroscopy, the Cameron erosion presented as a linear mucosal defect (white arrow). After partial gas desufflation the volume and diameter of the sliding hiatal hernia were reduced to provide a better presentation of the lesion and enable sampling. On the left middle and lower part of the image and on the lower right part of the image one can recognize several hematin spots. (B) Our patient’s Cameron erosion during the second (first follow-up) gastroscopy. The left half of the erosion (white arrow) is covered by fibrin, and the right half (black arrow) showed inflammatory alterations (microscopically red mucosa). There are several hematin spots on the middle and lower right side of the image. (C) Image from the last (second follow-up) gastroscopy with maximal gas insufflation. The Cameron erosion is resolved. (D) Image from the last (second follow-up) gastroscopy with maximal gas insufflation. The Cameron erosion is resolved. Figure 2. (A) Microscopic image (HE, magnification ×100) from the second tissue sampling: oxyntocardiac mucosa with minimal foveolar hyperplasia. HE: Hematoxylin and eosin staining. (B) Microscopic image (HE, magnification ×200) from the second tissue sampling: focal minor active inflammatory infiltrates in lamina propria with a slight edema and dilated capillaries. No ischemic stigmata were present. (C) Microscopic image (CAB, magnification x100) from the second tissue sampling: a minimal fibrosis in lamina propria in a single localized area. HP was not detectable. CAB – chromotrope aniline blue staining; HP – Helicobacter pylori. Table 1. Laboratory test results of our patient. The tests were performed at the time of the initial, first, and second follow-up gastroscopies. Unit of measurement Initial gastroscopy First follow-up gastroscopy Second follow-up gastroscopy Normal values WBC G/L 6.95 5.86 4.19 4.00–9.00 Platelets G/L 251 221 270 150–400 RBC T/L 4.43 4.55 4.60 3.80–5.50 Hemoglobin g/dL 14.9 15.0 14.8 12.0–16.5 Hematocrit % 40.3 43.1 41.3 36.0–48.0 MCH pg 33.7 32.9 32.2 27.0–36.0 MCV fL 90.9 94.8 89.9 80.0–101.0 MCHC g/dL 37.1 34.8 35.9 30.0–38.0 RDW-CV % 12.2 12.7 13.9 11.5–15.5 PTT % 96 100 94 70.0–130.0 INR 1.0 1.0 1.0 aPTT sec. 26 25 25 22–35 Fibrinogen g/L 2.99 2.96 3.14 1.80–4.50 TSH mcU/mL 0.91 1.53 0.93 0.36–3.60 CRP mg/L Negative Negative Negative 0–3 K mmol/L 4.6 3.4 3.6 3.5–5.4 Na mmol/L 141 139 139 132–148 Cl mmol/L 107 105 105 95–110 Ca mmol/L 2.3 2.3 2.3 2.0–2.8 Mg mmol/L 1.0 1.0 1.0 0.6–1.1 Creatinine mg/dL 0.7 0.7 0.8 0.5–1.1 eGFR ml/min/l >70 >70 >70 70–150 BUN mg/dL 13 13 12 7–18 Uric acid mg/dL 4.4 4.2 5.2 2.6–6.0 ASAT U/L 21 17 23 0–31 ALAT U/L 20 21 28 0–34 GGT U/L 18 21 22 0–38 AP U/L 125 135 119 50–135 Bilirubin mg/dL 0.9 1.1 1.3 0.2–1.1 Lipase U/L 116 105 106 73–393 Alpha-amylase U/L 53 55 48 25–115 Fe mcg/dL 115 190 119 50–170 Transferrin g/L 2.29 2.41 2.49 2.00–3.60 Transf. sat. % 36 56 34 15–55 Ferritin mcg/L 63 47 57 22–250 Glucose (NF) mg/dl 87 87 97 60–99 WBC – white blood cells; RBC – red blood cells; MCH – mean cell hemoglobin; MCV – mean cell volume; MCHC – mean cell hemoglobin concentration; RDW-CV – red blood cell distribution width; PT – prothrombin time; INR – international normalized ratio; aPTT – activated partial thromboplastin time; TSH – thyroid-stimulating hormone; CRP – C-reactive protein; K – potassium; Na – sodium; Cl – chloride; Ca – calcium; Mg – magnesium; eGFR – estimated glomerular filtration rate; BUN – blood urea nitrogen; ASAT – aspartate aminotransferase; ALAT – alanine aminotransferase; GGT – gamma-glutamyl transferase; AP – alkaline phosphatase; Fe – iron; Transf. sat. – transferrin saturation; NF – non-fasting. Table 2. Pathogeneses of Cameron lesions. Each factor is accompanied by a short explanation of its significance and role in the development of Cameron lesions. Commentary Presence of a diaphragmatic hernia (axial or paraesophageal) This is the most important factor. There are no Cameron lesions without a hiatal hernia. Larger hiatal hernias are associated with a greater risk of their occurrence (10–20% in axial hiatal hernias ≥5 cm) [4] Extraluminal rubbing of the upper stomach and lower esophagus against the diaphragmatic hiatus An important role is played by the permanent extraluminal rubbing of the upper stomach and lower esophagus against the diaphragmatic hiatus (the repeated ascending and descending movement associated with breathing and swallowing) and the intraluminal friction of the mucosal folds within the hiatal hernia against each other and at the level of the distal hernial ring. The gradient between the positive intraabdominal and negative intrathoracic pressure probably provokes an additional sliding movement of the gastro-esophageal junction between both cavities, resulting in mucosal distress with edema, petechiae, and ulcers. This effect is stronger in paraesophageal hiatal hernias Ratio between hernial opening and hernial size From the physical point of view, the ratio between the hernial opening (Hiatus oesophageus) and the hernial size appears to be most important. A significant trauma should be expected in smaller hiatal hernias and a greater friction in larger hernias [5]. The configuration of a hiatal hernia may change with time, which is why erosions or ulcers may appear at different locations [6] Dual-hit hypothesis Multiple studies showed that the mechanical trauma from outside is insufficient to cause mucosal defects. Cameron lesions occur only when the external compression on the gastric wall is combined with an aggressive influence from inside. According to the so-called ‘dual-hit hypothesis’ additional intraluminal and mucosal factors also play a substantial role: gastric acid, gastro-esophageal reflux, and the oral intake of NSAID for 3 days/week within a month. One study demonstrated that in large hiatal hernias, intraluminal lesions occur only when the gastro-esophageal flap valve is intact. This valve counteracts the reflux and affects the role of gastric acid [7,8] Helicobacter pylori There is no clear correlation between HP and Cameron lesions. Only 1/3 of all patients with Cameron lesions are HP-positive [9–11] Gastric ischemia and stasis Additional independent factors for the occurrence of Cameron lesions could be the so-called gastric ‘ischemia’ and ‘stasis’. In some patients, the diaphragmatic hiatus could be narrow enough to cause an intermittent venous stasis in the gastric wall when the diaphragm contracts (e.g., sneezing, coughing, choking). The pathogenesis of Cameron lesions is multifactorial and probably comprises patient’s genotype and phenotype, their comorbidities, and the oral intake of drugs. Patients with Cameron lesions often take iron supplements because the gastric stasis tablets and secretions can remain longer in the herniated stomach and cause pressure erosions [12,13] NSAID – Non-steroidal anti-inflammatory drugs; HP – Helicobacter pylori.	1 DOSAGE FORM, QID	DrugDosageText	CC BY-NC-ND	33473099	19,008,761	2021-01-21
What was the outcome of reaction 'Gastric ulcer'?	A 61-Year-Old Woman with Chronic Iron-Deficiency Anemia Due to a Cameron Lesion and a Response to Oral Application of Combined Poloxamer 407 with Hyaluronic Acid and Chondroitin Sulfate Following Single Treatment with Pantoprazole: A Case Report. BACKGROUND Cameron lesions are linear erosions and ulcers on the crests of gastric mucosal folds in the neck of a hiatal hernia and can be difficult to diagnose and treat. This report is of a case of chronic iron deficiency in a 61-year-old woman with a late diagnosis of a Cameron lesion, who did not respond to a single treatment with the proton pump inhibitor (PPI) pantoprazole, but was then treated with oral poloxamer 407 with hyaluronic acid and chondroitin sulfate in addition to PPI. CASE REPORT We report the case of a 61-year-old women with recurrent iron-deficiency anemia, first diagnosed 40 years prior to her presentation at our Endoscopy Unit, and an ongoing melena. We discovered an intrahiatal gastric mucosal defect, which we at first treated with proton pump inhibitors and sucralfate. After a follow-up gastroscopy revealed the persistence of the lesion, we decided to incorporate into the treatment a gel-like substance containing, among others, hyaluronic acid and chondroitin sulfate, and observed that the lesion resolved completely. CONCLUSIONS This report highlights that Cameron lesions should be considered in patients with hiatal hernia who have iron-deficiency anemia and can be diagnosed on upper endoscopy. Further clinical studies are required to determine the role of combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in the management of Cameron lesions. Background In 1986, Cameron and Higgins described linear erosions and ulcers on the crests of gastric mucosal folds in the neck of a hiatal hernia, resulting in gastrointestinal hemorrhage [1]. There is insufficient information on Cameron lesions in the published literature. The scarce data available are from single case reports or case series. Unfortunately, there are no randomized controlled studies on this topic. It is no exaggeration to mention that most textbooks in the field of internal medicine and surgery pay no attention to Cameron lesions, which is a fact that we will probably have to change soon. On the one hand, this is due to the small number of ‘Cameron’ patients worldwide and on the other hand because health care specialists do not recognize them. Even if recognized, a tissue sample from Cameron erosions or ulcers is rarely obtained, which explains the insufficient information about their histopathologic characteristics. We report the case of a 61-year-old woman with chronic iron-deficiency with a late diagnosis of a Cameron lesion, who did not respond to a single treatment with the proton pump inhibitor (PPI) pantoprazole, but was then treated orally with combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in addition to PPI. Case Report A 61-year-old woman presented for an outpatient gastroscopy due to ongoing melena. She took no iron supplementation or drugs that could have changed the stool color. The patient reported no abdominal or defecation problems. Previous surgeries included open appendectomy. The patient had psoriasis vulgaris as a single comorbidity and was a non-smoker. A prophylactic treatment with acetylsalicylic acid (ASA) was applied until 2009 because she had a stroke approximately 2 decades ago (the patient provided no medical records verifying this diagnosis). Two previous gastroscopies (the first at age 21 years and the second approximately 15 years ago) had already been performed because of a chronic ‘intermittent’ iron-deficiency anemia which was first diagnosed about 40 years ago. Both revealed no pathologies. A colonoscopy had also already been performed, with no detectable bleeding sources. After the above-mentioned uneventful endoscopic examinations, the treating physicians attributed the iron deficiency to the excessive menstrual bleeding our patient used to have and prescribed her iron supplementation. Unfortunately, the anemia persisted after menopause. The patient had not taken any iron supplements for 5 years prior to our gastroscopy. Having in mind this medical history, we ordered blood tests. The results are shown in Table 1. During our first gastroscopy, after an unproblematic esophageal intubation, we detected an axial hiatal hernia spreading approximately from 37 to 38 cm from the incisors, with a diameter of 2.2 cm. Advancing aborally, we saw multiple small, flat hematin spots which covered the gastric fundus and corpus, and were not present in the antral area. The duodenum was macroscopically intact. The decisive diagnostic factor proved to be the inversion of the fibroelastic gastroscope, as a small macroscopically visible, not bleeding linear erosion within the hiatal hernia (Figure 1A) was detected. Tissue samples were collected, which revealed only signs of carditis. A therapy with the proton pump inhibitors (PPI) pantoprazole and sucralfate was initiated for 6 weeks (pantoprazole 40 mg b.i.d. for 2 weeks, and afterwards once daily; sucral-fate 4 times daily) and an appointment for a follow-up gastroscopy was made. At the time of the follow-up gastroscopy (6 weeks later), the patient had no complaints and no melena, and all laboratory values were within reference ranges (Table 1). During the endoscopic examination we observed, once more, multiple flat hematin spots in the proximal two-thirds of the stomach. The erosion was still visible, although half of it was covered by fibrin (Figure 1B). We obtained tissue samples from the erosion. We changed the treatment to PPI (pantoprazole 40 mg once daily) and a gel containing, among others, hyaluronic acid, chondroitin sulfate, and poloxamer 407 (4 times daily, 30 min postprandially, and at bedtime) for 3 months. After the second gastroscopy, a new follow-up endoscopic appointment was made, which could not be kept due to the coronavirus crisis. We contacted the patient on the phone. She had no complaints and no melena, and after the above-mentioned 3-month period took only pantoprazole 40 mg once daily. Eventually, a second follow-up gastroscopy was performed 7 months after the initial one. It revealed no erosion, with an intact cardiac mucosa (Figure 1C, 1D). Discussion Our patient had a Cameron erosion that manifested with recurrent anemia and melena. After not achieving a full recovery under the most commonly described treatment with PPI, we decided to incorporate a combination of hyaluronic acid (HA), chondroitin sulfate (CS), and poloxamer 407 (in addition to PPI) into the therapy, and observed that the Cameron erosion disappeared. Cameron lesions are gastric mucosal defects of varying sizes localized within hiatal hernias. They were first described by the American physicians Alan J. Cameron and John A. Higgins (Mayo Clinic, 1986), who found a correlation between diaphragmatic hernias and occult gastrointestinal bleeding. The prevalence of Cameron lesions depends on 2 factors: the size of the hiatal hernia and the size of the hernial sac. Larger hernias are more frequently associated with the development of mucosal defects. Statistically, Cameron lesions are found in 5.2% of all patients with hiatal hernias undergoing gastroscopy, as more than 60% of them present with a single tissue defect. They are diagnosed in up to 50% of all gastroscopic examinations performed for other reasons [2–4]. Etiology and pathophysiology Cameron lesions are a complex phenomenon with an unclear pathogenesis. It is believed that they occur because of the combined effects of extra- and intraluminal mechanical and chemical factors. Pathogenetic factors are briefly presented in Table 2. Morphology Cameron lesions are non-peptic non-GERD-associated mucosal defects occurring on the top of gastric folds over the distal ring of a hiatal hernia (rarely proximally), most often along the lesser gastric curvature. They can be superficial (erosions) or deep (‘riding’ ulcers). Cameron lesions are typically longitudinal, but can also be oblong or ellipsoid, and never comprise the whole circumference of the hiatal hernia [14,15]. Symptoms Most patients with Cameron lesions are older women with chronic anemia and large hiatal hernias. Since hiatal hernias are predominantly asymptomatic, they present with signs of overt (melena, hematochezia, hematemesis) or occult (iron-deficiency anemia, positive fecal occult blood test) gastrointestinal bleeding. Some patients have a medical history of recurrent gastrointestinal bleeding, with or without blood transfusions, oral intake of NSAID, or iron supplements. Rarely, they also have a peptic ulcer or esophagitis [4]. Diagnostics The criterion standard for the diagnosis of Cameron lesions is gastroscopy. Very often, they remain uncovered within an index gastroscopy and are detected during a second-look or repeated gastroscopy (“One only sees what one looks for. One only looks for what one knows.” Goethe). That is why each hiatal hernia should be inspected ante- and retrograde with an additional perpendicular presentation of the hernial opening where the diaphragmatic hiatus exerts the highest pressure on the gastric wall (the predominant localization of Cameron ulcers). The gastroscopist should examine the mucosa above and beneath the Hiatus oesophageus, since Cameron lesions can migrate upwards or downwards depending on the extent to which the stomach is insufflated. The use of chromoendoscopy and optical magnification can also be helpful. Accompanying pathological findings are sometimes observed, such as mucosal edema, erythematous stomach lining, and ecchymoses on gastric folds. There are no specific laboratory tests or imaging modalities for detecting the presence of a Cameron lesion. Blood tests could be normal or demonstrate an iron-deficiency anemia. X-ray or computed tomography may be helpful for the diagnostics of complications (e.g., perforation, volvulus, blood clots in the stomach). Under certain conditions, computed tomography angiogram or Tc-99m-labeled red blood cells scintigraphy can detect an active bleeding source (bleeding speed at least 0.5 ml/min and 0.04 ml/min, respectively). The role of capsule endoscopy remains unclear [12,14]. Histopathologic characteristics To the best of our knowledge, there is only 1 published article containing a description of histopathologic changes in Cameron lesions. Katz et al. describe mucosal alterations due to mucosal vascular obstruction consistent with ischemic gastropathy, such as hemorrhagic infiltrates, fibrin thrombi, inflammatory response, sanguine micro-suffusions, sloughing of epithelial cells, atrophy of crypts, and coagulation necrosis [16]. Our first tissue sampling revealed mucosal alterations consistent with a minor chronic carditis. The results from the second sampling are shown in Figure 2. Management The treatment of Cameron lesions (conservative or surgical) should be determined individually [3]. Because of the limited knowledge about their natural history and the small number of patients, modern medicine was unable to create strict guidelines and algorithms for their management. Literature reports range from cases of spontaneous healing to high-dose PPI treatment regimens [8]. The management of Cameron lesions depends on their clinical presentation. Currently, most authors recommend starting PPI administration as soon as the diagnosis is established. If the patient already receives PPIs once daily, their dosage/frequency of administration should be increased. If blood tests show signs of iron deficiency, iron supplementation is required. In case it has already been initiated, the iron dosage should be increased. A single iron supplementation is not sufficient [17]. By means of the above-mentioned treatment, an adequate symptom control is achieved in up to half of the patients with Cameron lesions. Of course, in case of significant bleeding and hemodynamic shock, stabilizing measures should be taken, since Cameron lesions cause life-threatening gastrointestinal bleeding in up to one-third of all patients [11,18]. NSAID intake should be immediately discontinued. Some authors recommend the administration of prokinetic agents [19]. There are no strict criteria for the conversion from conservative to surgical treatment. Some specialists suggest a secondary surgical treatment in case of persistent complaints (e.g., anemia, hospitalization because of recurrent gastrointestinal bleeding or chronic blood loss) or complications (e.g., refractory bleeding, perforation, volvulus, gastric incarceration) [4]. The main goal of the surgical therapy is to eliminate the mandatory prerequisite for the occurrence of Cameron lesions – the hiatal hernia (there is no Cameron lesion without hiatal hernia.) Possible options are laparoscopic/open fundoplication±gastropexy and the recently developed single-incision transgastric underrunning (SILT) [3,9,20,21]. Endoscopy Endoscopic hemostasis is a relevant but rarely reported non-surgical treatment option for bleeding Cameron lesions. Band ligation and clipping proved to be effective. A follow-up gastroscopy was not performed in many studies, but could be indicated when complaints persist. Our patient After diagnosis establishment, we started a treatment with oral intake of PPIs (pantoprazole 40 mg b.i.d. for 2 weeks and once daily afterwards) in combination with sucralfate 4 times daily, both for 6 weeks. Since the iron panel revealed normal values, no iron supplementation was initiated. PPIs administration aims to inhibit gastric acid production; thus, its possible role in the genesis of Cameron lesions. Sucralfate is a sucrose octasulfate and aluminum hydroxide (AlOH) complex forming a viscous paste in aqueous acidic media (e.g., in the esophagus, stomach, duodenum). It adheres to defective and intact mucosa through polyvalent bridges between negatively charged sucralfate polyanions and positively charged proteins, which are present in high concentrations in mucosal lesions. It also buffers the gastric acid, inhibits the action of pepsin, and absorbs bile salts. All these properties enable sucralfate to act as an effective barrier counteracting the penetration of the above-mentioned substances. Sucralfate proved to be as effective as antacids or H2-receptor antagonists in healing peptic ulcers and in animals, but was ineffective in preventing corticosteroid-induced ulcerations. Sucralfate probably stimulates the local prostaglandin E2 (PGE2) and prostacyclin (PGI2) synthesis in gastric mucosa [22,23]. Since the first follow-up gastroscopy showed no improvement, we decided to use a mixed preparation of hyaluronic acid, chondroitin sulfate, and poloxamer 407. In animal models (Yorkshire pigs), this mixture exhibited positive effects on the healing process of scars left after endoscopic mucosal re-section (EMR) or endoscopic submucosal dissection (ESD) and had been expected to provoke an early proliferation of collagen and elastic fibers in gastric mucosal defects, thus also contributing to ulcer healing. Furthermore, it is an acceptable low-cost alternative to the existing treatment regimens [24]. Hyaluronic acid and chondroitin sulfate are extensively used in wound treatment. Their role in the therapy of gastrointestinal lesions is a subject of continuous research. Hyaluronic acid is a non-sulfated natural glycosaminoglycan and main component of the extracellular matrix. HA is a hygroscopic macromolecule formed by the polymerization of glucuronic acid and N-acetylglucosamine disaccharide. It interacts with several cell surface receptors of malignant and non-malignant gastric mucosa. HA is involved in human innate immunity and inflammatory processes, as it takes part in leukocyte recruitment and macrophage activation. It induces dendritic cell maturation and promotes cytokine release by dendritic cells and endothelial cells. HA also possesses anti-bacterial, anti-fungal, and anti-viral properties and promotes angiogenesis. Topically applied on oral mucosa, it supports tissue hydration in inflammatory processes and mucosal response to tissue injuries that could possibly result in ulcer formation and forms a protective film over exposed mucosal nerve endings, preventing them from overstimulation. Sodium hyaluronate possesses also hemostatic properties when injected in peptic ulcers. Chondroitin sulfate is a glycosaminoglycan consisting of repeated disaccharide units polymerized into long chains. CS is an effective inhibitor of pepsin-induced damage to stomach and duodenal mucosa and showed potent anti-inflammatory properties in animal models [25–27]. Poloxamer 407 is a water-soluble, non-ionic triblock copolymer that is liquid at room temperature and assumes a gel form at body temperature. It is often used as a drug carrier. It possesses mucoadhesive properties and does not irritate mucosal membranes. Poloxamer 407 showed no direct regenerative or antimicrobial properties but maintains a stable concentration of the substances it carries for a prolonged time [28]. Conclusions Hyaluronic acid and chondroitin sulfate are widely used in cosmetics because of their skin regeneration properties. They also possess a proven beneficial effect on the healing of acute and chronic wounds. The role of HA and CS in the treatment of gastrointestinal mucosal pathologies has increasingly been examined over the last decade. In accordance with the published literature and the experience with our patient, we hypothesize that they could also be useful for the conservative treatment of Cameron lesions (in combination with PPI, as stated in the summary of product characteristics of the products approved for use in the gastrointestinal tract). They counteract many of the possible organic and chemical pathogenetic factors, leading to its occurrence, but, of course, cannot influence the most important mechanical factor – the hiatal hernia. In light of the good success of HA and CS in wound healing, we hypothesize the principle that internal wounds (ulcers and mucosal lesions) could be treated like external wounds – treat ulcers like wounds (treat inside like outside). If not achieving a permanent resolution, it could at least gain us some time before surgery to stabilize the patient’s condition, which could sometimes be crucial. This case report highlights that Cameron lesions should be considered in patients with hiatal hernia who have iron-deficiency anemia and can be diagnosed on upper endoscopy. Further clinical studies are required to determine the role of combined poloxamer 407 with hyaluronic acid and chondroitin sulfate in the management of Cameron lesions. The authors would like to express their sincere gratitude to the teams of the Endoscopy Unit, Institute of Pathology, and the Department of General, Visceral and Vascular Surgery of the Steyr Regional Hospital for their help in obtaining and processing all the materials necessary for this article. Conflicts of interest None. Figure 1. (A) Our patient’s Cameron erosion at the time of diagnosis establishment. During the initial gastroscopy, the Cameron erosion presented as a linear mucosal defect (white arrow). After partial gas desufflation the volume and diameter of the sliding hiatal hernia were reduced to provide a better presentation of the lesion and enable sampling. On the left middle and lower part of the image and on the lower right part of the image one can recognize several hematin spots. (B) Our patient’s Cameron erosion during the second (first follow-up) gastroscopy. The left half of the erosion (white arrow) is covered by fibrin, and the right half (black arrow) showed inflammatory alterations (microscopically red mucosa). There are several hematin spots on the middle and lower right side of the image. (C) Image from the last (second follow-up) gastroscopy with maximal gas insufflation. The Cameron erosion is resolved. (D) Image from the last (second follow-up) gastroscopy with maximal gas insufflation. The Cameron erosion is resolved. Figure 2. (A) Microscopic image (HE, magnification ×100) from the second tissue sampling: oxyntocardiac mucosa with minimal foveolar hyperplasia. HE: Hematoxylin and eosin staining. (B) Microscopic image (HE, magnification ×200) from the second tissue sampling: focal minor active inflammatory infiltrates in lamina propria with a slight edema and dilated capillaries. No ischemic stigmata were present. (C) Microscopic image (CAB, magnification x100) from the second tissue sampling: a minimal fibrosis in lamina propria in a single localized area. HP was not detectable. CAB – chromotrope aniline blue staining; HP – Helicobacter pylori. Table 1. Laboratory test results of our patient. The tests were performed at the time of the initial, first, and second follow-up gastroscopies. Unit of measurement Initial gastroscopy First follow-up gastroscopy Second follow-up gastroscopy Normal values WBC G/L 6.95 5.86 4.19 4.00–9.00 Platelets G/L 251 221 270 150–400 RBC T/L 4.43 4.55 4.60 3.80–5.50 Hemoglobin g/dL 14.9 15.0 14.8 12.0–16.5 Hematocrit % 40.3 43.1 41.3 36.0–48.0 MCH pg 33.7 32.9 32.2 27.0–36.0 MCV fL 90.9 94.8 89.9 80.0–101.0 MCHC g/dL 37.1 34.8 35.9 30.0–38.0 RDW-CV % 12.2 12.7 13.9 11.5–15.5 PTT % 96 100 94 70.0–130.0 INR 1.0 1.0 1.0 aPTT sec. 26 25 25 22–35 Fibrinogen g/L 2.99 2.96 3.14 1.80–4.50 TSH mcU/mL 0.91 1.53 0.93 0.36–3.60 CRP mg/L Negative Negative Negative 0–3 K mmol/L 4.6 3.4 3.6 3.5–5.4 Na mmol/L 141 139 139 132–148 Cl mmol/L 107 105 105 95–110 Ca mmol/L 2.3 2.3 2.3 2.0–2.8 Mg mmol/L 1.0 1.0 1.0 0.6–1.1 Creatinine mg/dL 0.7 0.7 0.8 0.5–1.1 eGFR ml/min/l >70 >70 >70 70–150 BUN mg/dL 13 13 12 7–18 Uric acid mg/dL 4.4 4.2 5.2 2.6–6.0 ASAT U/L 21 17 23 0–31 ALAT U/L 20 21 28 0–34 GGT U/L 18 21 22 0–38 AP U/L 125 135 119 50–135 Bilirubin mg/dL 0.9 1.1 1.3 0.2–1.1 Lipase U/L 116 105 106 73–393 Alpha-amylase U/L 53 55 48 25–115 Fe mcg/dL 115 190 119 50–170 Transferrin g/L 2.29 2.41 2.49 2.00–3.60 Transf. sat. % 36 56 34 15–55 Ferritin mcg/L 63 47 57 22–250 Glucose (NF) mg/dl 87 87 97 60–99 WBC – white blood cells; RBC – red blood cells; MCH – mean cell hemoglobin; MCV – mean cell volume; MCHC – mean cell hemoglobin concentration; RDW-CV – red blood cell distribution width; PT – prothrombin time; INR – international normalized ratio; aPTT – activated partial thromboplastin time; TSH – thyroid-stimulating hormone; CRP – C-reactive protein; K – potassium; Na – sodium; Cl – chloride; Ca – calcium; Mg – magnesium; eGFR – estimated glomerular filtration rate; BUN – blood urea nitrogen; ASAT – aspartate aminotransferase; ALAT – alanine aminotransferase; GGT – gamma-glutamyl transferase; AP – alkaline phosphatase; Fe – iron; Transf. sat. – transferrin saturation; NF – non-fasting. Table 2. Pathogeneses of Cameron lesions. Each factor is accompanied by a short explanation of its significance and role in the development of Cameron lesions. Commentary Presence of a diaphragmatic hernia (axial or paraesophageal) This is the most important factor. There are no Cameron lesions without a hiatal hernia. Larger hiatal hernias are associated with a greater risk of their occurrence (10–20% in axial hiatal hernias ≥5 cm) [4] Extraluminal rubbing of the upper stomach and lower esophagus against the diaphragmatic hiatus An important role is played by the permanent extraluminal rubbing of the upper stomach and lower esophagus against the diaphragmatic hiatus (the repeated ascending and descending movement associated with breathing and swallowing) and the intraluminal friction of the mucosal folds within the hiatal hernia against each other and at the level of the distal hernial ring. The gradient between the positive intraabdominal and negative intrathoracic pressure probably provokes an additional sliding movement of the gastro-esophageal junction between both cavities, resulting in mucosal distress with edema, petechiae, and ulcers. This effect is stronger in paraesophageal hiatal hernias Ratio between hernial opening and hernial size From the physical point of view, the ratio between the hernial opening (Hiatus oesophageus) and the hernial size appears to be most important. A significant trauma should be expected in smaller hiatal hernias and a greater friction in larger hernias [5]. The configuration of a hiatal hernia may change with time, which is why erosions or ulcers may appear at different locations [6] Dual-hit hypothesis Multiple studies showed that the mechanical trauma from outside is insufficient to cause mucosal defects. Cameron lesions occur only when the external compression on the gastric wall is combined with an aggressive influence from inside. According to the so-called ‘dual-hit hypothesis’ additional intraluminal and mucosal factors also play a substantial role: gastric acid, gastro-esophageal reflux, and the oral intake of NSAID for 3 days/week within a month. One study demonstrated that in large hiatal hernias, intraluminal lesions occur only when the gastro-esophageal flap valve is intact. This valve counteracts the reflux and affects the role of gastric acid [7,8] Helicobacter pylori There is no clear correlation between HP and Cameron lesions. Only 1/3 of all patients with Cameron lesions are HP-positive [9–11] Gastric ischemia and stasis Additional independent factors for the occurrence of Cameron lesions could be the so-called gastric ‘ischemia’ and ‘stasis’. In some patients, the diaphragmatic hiatus could be narrow enough to cause an intermittent venous stasis in the gastric wall when the diaphragm contracts (e.g., sneezing, coughing, choking). The pathogenesis of Cameron lesions is multifactorial and probably comprises patient’s genotype and phenotype, their comorbidities, and the oral intake of drugs. Patients with Cameron lesions often take iron supplements because the gastric stasis tablets and secretions can remain longer in the herniated stomach and cause pressure erosions [12,13] NSAID – Non-steroidal anti-inflammatory drugs; HP – Helicobacter pylori.	Recovered	ReactionOutcome	CC BY-NC-ND	33473099	19,008,761	2021-01-21
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Dystrophic calcification'.	Delayed Bladder Perforation Related to Immediate Single Dose Intravesical Doxorubicin Instillation After TURBT: A Case Report and Literature Review. Intravesical chemotherapy instillation immediately after tumor resection is a well-known practice in the management of non-muscle invasive bladder cancer. Despite being largely well tolerated in most cases, it is not devoid of severe and life-threatening complications. We present an unusual case of bladder perforation that happened 2 weeks after bladder tumor resection. The patient had received single dose intra-vesical instillation of doxorubicin after TUR-BT. Conservative managements failed to achieve bladder healing; as a result, open surgical repair was performed. To the best of our knowledge, this is the first reported case of bladder perforation after intra-vesical doxorubicin instillation. The occurrence of such a rare serious complication in a mostly safe intervention must be taken into consideration. A high index of suspicion, timely management, and proceeding to more invasive surgical treatments when necessary are cornerstones in the management and preserving the bladder. Introduction Bladder cancer (BC) is the seventh most commonly diagnosed cancer in men worldwide. About 75% of the cases are Non-muscle invasive (NMIBC).1 Transurethral resection of the bladder tumor (TURBT) remains the gold standard of therapy for diagnosing and treating NMIBC. To decrease the recurrence rate of low-risk tumors, a single, immediate, post-operative intravesical instillation of chemotherapy is recommended.1 The most commonly used chemotherapeutic agent is Mitomycin C (MMC), which is an alkylating agent that inhibits DNA synthesis and causes single-strand breakage of DNA and chromosomal breaks.2 Immediate single instillation (SI) is largely well-tolerated. However, severe complications and even death have been reported in the literature. Here we present a case wherein the patient had an extraperitoneal bladder perforation that occurred 2 weeks after TUT-BT and immediate postoperative instillation of doxorubicin. Case Presentation A 63-Year-old male, a heavy smoker, with no other co-morbidities, was referred to urology clinic for evaluation and management of a lesion on the left bladder wall that was incidentally detected on a CT scan. He did not have any urinary symptoms on initial presentation. Cystourethroscopy revealed 2 papillary growths on the left lateral bladder wall, which were resected completely and smoothly by a resectoscope using a monopolar energy. Complete resection of the tumor was achieved without any intra-operative complication or perforation. Hemostasis was secured, and a urethral catheter was inserted. In accordance of the EAU guidelines, the patient received an intravesical instillation of 50 ml of doxorubicin diluted in 100 cc of saline 6 hours after the TURBT. During the instillation, his vital signs were stable and he had neither hematuria nor abdominal pain. One hour after the instillation, the catheter was unclamped and was attached to continuous bladder irrigation. Two days after the surgery and in the absence of hematuria, the indwelling catheter was removed, and the patient was discharged home the next day. When seen in the clinic 1 week later the patient was fine with no alarming symptoms. The pathology report showed a low-grade Ta Papillary urothelial carcinoma. Based on the pathological diagnosis and according to the EAU guidelines, adjuvant chemotherapy instillations were planned. Two weeks later, the patient presented with, dysuria, frequency, urgency, as well as pelvic and perineal pain. Physical examination was unremarkable. Urine analysis showed numerous WBC’s, RBC’s, and bacteria. An empirical antibiotics course was commenced. In view of the worsening symptoms and the past history of anal fistula, a contrast enhanced pelvic MRI was performed. It showed a diffusely thickened urinary bladder wall, with evidence of a focal wall defect at its left aspect (the site of the resected tumor). The active urine leak into the perivesical space confirmed the diagnosis of retroperitoneal bladder perforation (Figure 1). Figure. 1. Pelvic MRI (axial view) showing extraperitoneal bladder perforation at the left lateral wall (arrow points to extravasated contrast). The patient was managed conservatively with antibiotics and an indwelling catheter for 3 weeks. A follow-up retrograde CT cystogram showed no improvement and a gross contrast leak from the same site was identified. The catheter was kept for 6 more weeks and the CT cystogram was repeated showing the same results with no evidence of improvement. The patient refused to keep the catheter for a longer period so the option of open surgical repair was discussed with him. Extraperitoneal laparotomy was performed, a perivesical small abscess was drained and cultured, necrotic tissues were debrided, and the bladder wall defect was rimed and closed in 3 layers. Tissue biopsies from the pelvic wall and bladder wall were harvested. A urethral catheter and pelvic drain were kept in place. The postoperative course was uneventful, the drain was removed on the third-day postoperatively, and the patient was discharged home with the urethral catheter in. Histopathological evaluation of the pelvic wall and the bladder biopsies verified acute and chronic inflammation as well as granulation tissue formation. However, both were negative for malignancy. The urinary bladder biopsy also showed extensive foci of dystrophic calcification while the pelvic wall biopsy revealed fat necrosis (Figures 2 and 3). Figure 2. The histologic examination revealed fragments of soft tissue with fat necrosis in the lower left area, mixed acute and chronic inflammatory cell infiltrate and hemorrhage in the upper right area (Hematoxylin & Eosin, 200X). Figure 3. The histologic examination revealed multiple areas of calcifications embedded deep in the tissue with associated crystal-like material suggestive of calcium-oxalate or talc powder crystals (Hematoxylin & Eosin, 200X). A follow up CT cystogram was done 3 weeks after the bladder repair. Although it showed a considerable regression in the size of the bladder defect, interestingly it also showed persistent extra-peritoneal contrast leak from the bladder along it’s left lateral aspect (Figure 4). Figure 4. Retrograde Ct cystogram showing persistent contrast leak from the left lateral aspect after surgical repair. Due to this slow healing, the catheter was kept in. Six weeks later, a follow up CT cystogram showed healing with no extraperitoneal contrast leak (Figure 5). And hence the catheter was removed. Figure 5. Retrograde Ct cystogram showing a healed bladder wall with no contrast leak. Two follow up cystoscopies and bladder biopsy (from an erythematous area on the left lateral wall) with 3 months intervals showed good healing of the bladder wall and no recurrence of the tumor. Discussion Single intravesical instillation with mitomycin C (MMC), epirubicin, or pirarubicin, have shown a beneficial effect in reducing recurrence rate compared to TURBT alone. However, randomized comparisons of individual drugs have not been conducted.3 The high recurrence rate of NMIBC may be attributed to the seeding of floating tumor cells. Immediate chemotherapy instillation has been shown to act by destroying these circulating tumor cells after TURBT. Hence the EAU guidelines recommend initiating the instillation within the first few hours after TURBT.1 Most of the complications of intravesical chemotherapy instillation are minor and can be managed medically. Dysuria and urinary frequency and urgency were the most common complications.4 On the other hand, reporting major complications such as bladder perforation, perirectal abscesses, eosinophilic cystitis, and ureteral stenosis is limited to case reports.5-14 In this case, we described our experience with bladder perforation after immediate intravesical instillation of doxorubicin. One can argue that the perforation has occurred intraoperatively and gone unnoticed. However, in view of the absence of endoscopic evidence of perforation, late presentation of symptoms, and the absence of symptoms during and after the instillation we assume that the Single Dose Intravesical Doxorubicin Instillation caused the perforation rather than complicating an existing one. To explain the mechanism of perforation, Lim et al7 and Cliff et al8 hypothesized that perforation is caused when attenuated muscularis propria gets necrosed after MMC instillation. The presenting complaints of our patient were perineal and pelvic pain as well as urinary irritative symptoms. This is similar to Elmamoun et al9 cohort in which all the 6 patients presented with perineal and pelvic pain. Other symptoms such as fever, urine retention, and recurrent UTI were variably reported by others. Table 1 summarizes the clinical presentation and management of bladder perforation in the previously published case reports. Table1. A summary of the presentation and management of bladder perforation after immediate chemotheraputic agent intravesical instillation. Ref. number The instilled drug Number of patients Initial presentation Management outline Lim et al7 MMC 1 Pelvic and suprapubic pain Exploratry laparotomy and bladder repair after failed conservative management Elmamoun et al9 MMC 6 3 patients presented with pelvic pain Conservative management and urethral catheterization for 2, 26, and 52 weeks Pelvic pain Recurrent UTIs Left ureteric obstruction Partial cystectomy + left to right trans-ureteroureterostomy Pelvic pain, Persistent urinary leak Left ureteric obstruction Augmentation cystoplasty + leftUreteric reimplantationThe patient ended with persistent leak, so a long-term catheter was used Pelvic painSevere LUTS Bilateral ureteric obstruction Urinary diversion (ileal conduit) Penna et al10 MMC 1 Lower abdominal pain and inability to void Analgesia, antibiotics and urethral catheter for 4 weeks Tyritzis et al11 EPI 2 Fever and retropubic pain Conservative: Antibiotics and urethral catheter Fever, lower abdominal pain and cellulitis Exploratry laparotomy and bladder repair after failed conservative management Nieuwenhuijzen et al12 MMC Pelvic pain and fever Exploratry laparotomy and bladder repair after failed conservative management for 1 month Racioppi et al13 MMC Pelvic pain, fever and pancytopenia Exploratry laparotomy and bladder repair after failed conservative management for 3 weeks Hatem and Leifeld14 MMC Pelvic pain Fever and positive blood culture for ecoli as well as purulent discharge from the drain site after the first expolratory laparatomy Conservative management for 10 daysExploratory laparatomy that revealed edematous anterior bladder wall without defectRenewed laparotomy, drainage of the abscess, debridement of the necrotic tissue, mono-J ureteral stents and nephrostomyRadical cystectomy with a urinary diversion through ureterocutaneostomy Shenaq et al15 EPI 3 Strong urge and pain in the lower left abdomen Conservative management with antibiotics and urethral catheterization Abdominal pain and intestinal obstruction Conservative management for 12 daysExploratory laparotomy that revealed infiltrative mass on the top of the bladder causing IO, so colostomy was done.The patient died due to multi organ failure Progressive pain in the lower abdomen Conservative management with antibiotics and urethral catheterization Abbreviations: Ep, epirubicin; MMC, mitomycin C. Concerning the management, almost all the reported cases were initially managed conservatively by urethral catheterization, anticholinergics, and antibiotics. However, this was insufficient to heal the bladder in some cases which needed open repair. Moreover, some ended with cystectomy. In our case, the patient has an extraperitoneal extravasation that failed the conservative management and needed open surgical repair. A striking feature of the healing process that was seen in many cases including ours is the poor and slow healing. In our case, the patient needed 9 weeks for complete healing after proper open 3 layers bladder wall repair.7-9,13,14 While Lim et al attributed the slow healing in their case to the patient’s pre-existing peripheral vascular disease and suboptimal tissue oxygenation, no risk factors were identified in the other cases including our patient who was free of any comorbidities. We suppose that the poor healing is linked to the chemotherapeutic agent inflammatory effect rather than to patient factor. This assumption is backed by the reported characteristic of mitomycin induced skin ulcer in cases of extravasation after intravenous administration. These ulcers are well known for their chronicity and the low tendency of spontaneous healing. Moreover, debridement and soft-tissue reconstruction following extravasation of MMC are recommended for patients with persistent ulceration and pain.15 Another common finding in the cases that mandate open repair was the presence of perivesical soft tissue necrosis and extensive inflammation. Doherty et al observed that more extensive bladder wall and fat necrosis of extravesical tissue in cystectomy specimens occurred when either epirubicin or mitomycin instillation was administered than in that seen following TURBT alone.16 This cytotoxic effect was also reported by Tyritzis et al11 after epirubicin instillation, and by Cliff et al,8 Nieuwenhuijzen et al,12 and Hatem and Leifeld14 after MMC instillation. This cytotoxic effect was recognized herein for the doxorubicin instillation, which is the first time to be reported as a complication of doxorubicin. It is worth to mention that the perforation had no adverse oncological effect on our patient as no tumor recurrence was detected during follow up. Conclusion Immediate intravesical instillations of chemotherapy are not devoid from risk and serious complications as bladder perforation can occur. Whether it’s value in decreasing the recurrence justify the risk of bladder perforation is not our argument in this case report. Nevertheless, this severe complication should be kept in mind, and high index of suspicion will aid in timely management. If perforation is suspected, urethral catheterization and contrasted imaging are mandatory. In the case of failed conservative management surgical repair is warranted even in extraperitoneal leak. Funding:The author(s) received no financial support for the research, authorship, and/or publication of this article. Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Author Contributions: Concept: AA, SAD. Design: MA, SA. Supervision: AA, SAD, MAA. Resources: MA, HM, NB. Materials: AA, MA, NB. Writing manuscript: AA, MA, HM. Critical review: AA, SAD, MAA. Informed Consent: The patient has given his written informed consent to publish his case including publication of images. The study follows the guidelines for human studies and is conducted in accordance with the World Medical Association Declaration of Helsinki. ORCID iD: Hammam Mansi https://orcid.org/0000-0002-4571-4336	DOXORUBICIN	DrugsGivenReaction	CC BY-NC	33473244	18,858,074	2021
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Dysuria'.	Delayed Bladder Perforation Related to Immediate Single Dose Intravesical Doxorubicin Instillation After TURBT: A Case Report and Literature Review. Intravesical chemotherapy instillation immediately after tumor resection is a well-known practice in the management of non-muscle invasive bladder cancer. Despite being largely well tolerated in most cases, it is not devoid of severe and life-threatening complications. We present an unusual case of bladder perforation that happened 2 weeks after bladder tumor resection. The patient had received single dose intra-vesical instillation of doxorubicin after TUR-BT. Conservative managements failed to achieve bladder healing; as a result, open surgical repair was performed. To the best of our knowledge, this is the first reported case of bladder perforation after intra-vesical doxorubicin instillation. The occurrence of such a rare serious complication in a mostly safe intervention must be taken into consideration. A high index of suspicion, timely management, and proceeding to more invasive surgical treatments when necessary are cornerstones in the management and preserving the bladder. Introduction Bladder cancer (BC) is the seventh most commonly diagnosed cancer in men worldwide. About 75% of the cases are Non-muscle invasive (NMIBC).1 Transurethral resection of the bladder tumor (TURBT) remains the gold standard of therapy for diagnosing and treating NMIBC. To decrease the recurrence rate of low-risk tumors, a single, immediate, post-operative intravesical instillation of chemotherapy is recommended.1 The most commonly used chemotherapeutic agent is Mitomycin C (MMC), which is an alkylating agent that inhibits DNA synthesis and causes single-strand breakage of DNA and chromosomal breaks.2 Immediate single instillation (SI) is largely well-tolerated. However, severe complications and even death have been reported in the literature. Here we present a case wherein the patient had an extraperitoneal bladder perforation that occurred 2 weeks after TUT-BT and immediate postoperative instillation of doxorubicin. Case Presentation A 63-Year-old male, a heavy smoker, with no other co-morbidities, was referred to urology clinic for evaluation and management of a lesion on the left bladder wall that was incidentally detected on a CT scan. He did not have any urinary symptoms on initial presentation. Cystourethroscopy revealed 2 papillary growths on the left lateral bladder wall, which were resected completely and smoothly by a resectoscope using a monopolar energy. Complete resection of the tumor was achieved without any intra-operative complication or perforation. Hemostasis was secured, and a urethral catheter was inserted. In accordance of the EAU guidelines, the patient received an intravesical instillation of 50 ml of doxorubicin diluted in 100 cc of saline 6 hours after the TURBT. During the instillation, his vital signs were stable and he had neither hematuria nor abdominal pain. One hour after the instillation, the catheter was unclamped and was attached to continuous bladder irrigation. Two days after the surgery and in the absence of hematuria, the indwelling catheter was removed, and the patient was discharged home the next day. When seen in the clinic 1 week later the patient was fine with no alarming symptoms. The pathology report showed a low-grade Ta Papillary urothelial carcinoma. Based on the pathological diagnosis and according to the EAU guidelines, adjuvant chemotherapy instillations were planned. Two weeks later, the patient presented with, dysuria, frequency, urgency, as well as pelvic and perineal pain. Physical examination was unremarkable. Urine analysis showed numerous WBC’s, RBC’s, and bacteria. An empirical antibiotics course was commenced. In view of the worsening symptoms and the past history of anal fistula, a contrast enhanced pelvic MRI was performed. It showed a diffusely thickened urinary bladder wall, with evidence of a focal wall defect at its left aspect (the site of the resected tumor). The active urine leak into the perivesical space confirmed the diagnosis of retroperitoneal bladder perforation (Figure 1). Figure. 1. Pelvic MRI (axial view) showing extraperitoneal bladder perforation at the left lateral wall (arrow points to extravasated contrast). The patient was managed conservatively with antibiotics and an indwelling catheter for 3 weeks. A follow-up retrograde CT cystogram showed no improvement and a gross contrast leak from the same site was identified. The catheter was kept for 6 more weeks and the CT cystogram was repeated showing the same results with no evidence of improvement. The patient refused to keep the catheter for a longer period so the option of open surgical repair was discussed with him. Extraperitoneal laparotomy was performed, a perivesical small abscess was drained and cultured, necrotic tissues were debrided, and the bladder wall defect was rimed and closed in 3 layers. Tissue biopsies from the pelvic wall and bladder wall were harvested. A urethral catheter and pelvic drain were kept in place. The postoperative course was uneventful, the drain was removed on the third-day postoperatively, and the patient was discharged home with the urethral catheter in. Histopathological evaluation of the pelvic wall and the bladder biopsies verified acute and chronic inflammation as well as granulation tissue formation. However, both were negative for malignancy. The urinary bladder biopsy also showed extensive foci of dystrophic calcification while the pelvic wall biopsy revealed fat necrosis (Figures 2 and 3). Figure 2. The histologic examination revealed fragments of soft tissue with fat necrosis in the lower left area, mixed acute and chronic inflammatory cell infiltrate and hemorrhage in the upper right area (Hematoxylin & Eosin, 200X). Figure 3. The histologic examination revealed multiple areas of calcifications embedded deep in the tissue with associated crystal-like material suggestive of calcium-oxalate or talc powder crystals (Hematoxylin & Eosin, 200X). A follow up CT cystogram was done 3 weeks after the bladder repair. Although it showed a considerable regression in the size of the bladder defect, interestingly it also showed persistent extra-peritoneal contrast leak from the bladder along it’s left lateral aspect (Figure 4). Figure 4. Retrograde Ct cystogram showing persistent contrast leak from the left lateral aspect after surgical repair. Due to this slow healing, the catheter was kept in. Six weeks later, a follow up CT cystogram showed healing with no extraperitoneal contrast leak (Figure 5). And hence the catheter was removed. Figure 5. Retrograde Ct cystogram showing a healed bladder wall with no contrast leak. Two follow up cystoscopies and bladder biopsy (from an erythematous area on the left lateral wall) with 3 months intervals showed good healing of the bladder wall and no recurrence of the tumor. Discussion Single intravesical instillation with mitomycin C (MMC), epirubicin, or pirarubicin, have shown a beneficial effect in reducing recurrence rate compared to TURBT alone. However, randomized comparisons of individual drugs have not been conducted.3 The high recurrence rate of NMIBC may be attributed to the seeding of floating tumor cells. Immediate chemotherapy instillation has been shown to act by destroying these circulating tumor cells after TURBT. Hence the EAU guidelines recommend initiating the instillation within the first few hours after TURBT.1 Most of the complications of intravesical chemotherapy instillation are minor and can be managed medically. Dysuria and urinary frequency and urgency were the most common complications.4 On the other hand, reporting major complications such as bladder perforation, perirectal abscesses, eosinophilic cystitis, and ureteral stenosis is limited to case reports.5-14 In this case, we described our experience with bladder perforation after immediate intravesical instillation of doxorubicin. One can argue that the perforation has occurred intraoperatively and gone unnoticed. However, in view of the absence of endoscopic evidence of perforation, late presentation of symptoms, and the absence of symptoms during and after the instillation we assume that the Single Dose Intravesical Doxorubicin Instillation caused the perforation rather than complicating an existing one. To explain the mechanism of perforation, Lim et al7 and Cliff et al8 hypothesized that perforation is caused when attenuated muscularis propria gets necrosed after MMC instillation. The presenting complaints of our patient were perineal and pelvic pain as well as urinary irritative symptoms. This is similar to Elmamoun et al9 cohort in which all the 6 patients presented with perineal and pelvic pain. Other symptoms such as fever, urine retention, and recurrent UTI were variably reported by others. Table 1 summarizes the clinical presentation and management of bladder perforation in the previously published case reports. Table1. A summary of the presentation and management of bladder perforation after immediate chemotheraputic agent intravesical instillation. Ref. number The instilled drug Number of patients Initial presentation Management outline Lim et al7 MMC 1 Pelvic and suprapubic pain Exploratry laparotomy and bladder repair after failed conservative management Elmamoun et al9 MMC 6 3 patients presented with pelvic pain Conservative management and urethral catheterization for 2, 26, and 52 weeks Pelvic pain Recurrent UTIs Left ureteric obstruction Partial cystectomy + left to right trans-ureteroureterostomy Pelvic pain, Persistent urinary leak Left ureteric obstruction Augmentation cystoplasty + leftUreteric reimplantationThe patient ended with persistent leak, so a long-term catheter was used Pelvic painSevere LUTS Bilateral ureteric obstruction Urinary diversion (ileal conduit) Penna et al10 MMC 1 Lower abdominal pain and inability to void Analgesia, antibiotics and urethral catheter for 4 weeks Tyritzis et al11 EPI 2 Fever and retropubic pain Conservative: Antibiotics and urethral catheter Fever, lower abdominal pain and cellulitis Exploratry laparotomy and bladder repair after failed conservative management Nieuwenhuijzen et al12 MMC Pelvic pain and fever Exploratry laparotomy and bladder repair after failed conservative management for 1 month Racioppi et al13 MMC Pelvic pain, fever and pancytopenia Exploratry laparotomy and bladder repair after failed conservative management for 3 weeks Hatem and Leifeld14 MMC Pelvic pain Fever and positive blood culture for ecoli as well as purulent discharge from the drain site after the first expolratory laparatomy Conservative management for 10 daysExploratory laparatomy that revealed edematous anterior bladder wall without defectRenewed laparotomy, drainage of the abscess, debridement of the necrotic tissue, mono-J ureteral stents and nephrostomyRadical cystectomy with a urinary diversion through ureterocutaneostomy Shenaq et al15 EPI 3 Strong urge and pain in the lower left abdomen Conservative management with antibiotics and urethral catheterization Abdominal pain and intestinal obstruction Conservative management for 12 daysExploratory laparotomy that revealed infiltrative mass on the top of the bladder causing IO, so colostomy was done.The patient died due to multi organ failure Progressive pain in the lower abdomen Conservative management with antibiotics and urethral catheterization Abbreviations: Ep, epirubicin; MMC, mitomycin C. Concerning the management, almost all the reported cases were initially managed conservatively by urethral catheterization, anticholinergics, and antibiotics. However, this was insufficient to heal the bladder in some cases which needed open repair. Moreover, some ended with cystectomy. In our case, the patient has an extraperitoneal extravasation that failed the conservative management and needed open surgical repair. A striking feature of the healing process that was seen in many cases including ours is the poor and slow healing. In our case, the patient needed 9 weeks for complete healing after proper open 3 layers bladder wall repair.7-9,13,14 While Lim et al attributed the slow healing in their case to the patient’s pre-existing peripheral vascular disease and suboptimal tissue oxygenation, no risk factors were identified in the other cases including our patient who was free of any comorbidities. We suppose that the poor healing is linked to the chemotherapeutic agent inflammatory effect rather than to patient factor. This assumption is backed by the reported characteristic of mitomycin induced skin ulcer in cases of extravasation after intravenous administration. These ulcers are well known for their chronicity and the low tendency of spontaneous healing. Moreover, debridement and soft-tissue reconstruction following extravasation of MMC are recommended for patients with persistent ulceration and pain.15 Another common finding in the cases that mandate open repair was the presence of perivesical soft tissue necrosis and extensive inflammation. Doherty et al observed that more extensive bladder wall and fat necrosis of extravesical tissue in cystectomy specimens occurred when either epirubicin or mitomycin instillation was administered than in that seen following TURBT alone.16 This cytotoxic effect was also reported by Tyritzis et al11 after epirubicin instillation, and by Cliff et al,8 Nieuwenhuijzen et al,12 and Hatem and Leifeld14 after MMC instillation. This cytotoxic effect was recognized herein for the doxorubicin instillation, which is the first time to be reported as a complication of doxorubicin. It is worth to mention that the perforation had no adverse oncological effect on our patient as no tumor recurrence was detected during follow up. Conclusion Immediate intravesical instillations of chemotherapy are not devoid from risk and serious complications as bladder perforation can occur. Whether it’s value in decreasing the recurrence justify the risk of bladder perforation is not our argument in this case report. Nevertheless, this severe complication should be kept in mind, and high index of suspicion will aid in timely management. If perforation is suspected, urethral catheterization and contrasted imaging are mandatory. In the case of failed conservative management surgical repair is warranted even in extraperitoneal leak. Funding:The author(s) received no financial support for the research, authorship, and/or publication of this article. Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Author Contributions: Concept: AA, SAD. Design: MA, SA. Supervision: AA, SAD, MAA. Resources: MA, HM, NB. Materials: AA, MA, NB. Writing manuscript: AA, MA, HM. Critical review: AA, SAD, MAA. Informed Consent: The patient has given his written informed consent to publish his case including publication of images. The study follows the guidelines for human studies and is conducted in accordance with the World Medical Association Declaration of Helsinki. ORCID iD: Hammam Mansi https://orcid.org/0000-0002-4571-4336	DOXORUBICIN	DrugsGivenReaction	CC BY-NC	33473244	18,858,074	2021
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Extravasation of urine'.	Delayed Bladder Perforation Related to Immediate Single Dose Intravesical Doxorubicin Instillation After TURBT: A Case Report and Literature Review. Intravesical chemotherapy instillation immediately after tumor resection is a well-known practice in the management of non-muscle invasive bladder cancer. Despite being largely well tolerated in most cases, it is not devoid of severe and life-threatening complications. We present an unusual case of bladder perforation that happened 2 weeks after bladder tumor resection. The patient had received single dose intra-vesical instillation of doxorubicin after TUR-BT. Conservative managements failed to achieve bladder healing; as a result, open surgical repair was performed. To the best of our knowledge, this is the first reported case of bladder perforation after intra-vesical doxorubicin instillation. The occurrence of such a rare serious complication in a mostly safe intervention must be taken into consideration. A high index of suspicion, timely management, and proceeding to more invasive surgical treatments when necessary are cornerstones in the management and preserving the bladder. Introduction Bladder cancer (BC) is the seventh most commonly diagnosed cancer in men worldwide. About 75% of the cases are Non-muscle invasive (NMIBC).1 Transurethral resection of the bladder tumor (TURBT) remains the gold standard of therapy for diagnosing and treating NMIBC. To decrease the recurrence rate of low-risk tumors, a single, immediate, post-operative intravesical instillation of chemotherapy is recommended.1 The most commonly used chemotherapeutic agent is Mitomycin C (MMC), which is an alkylating agent that inhibits DNA synthesis and causes single-strand breakage of DNA and chromosomal breaks.2 Immediate single instillation (SI) is largely well-tolerated. However, severe complications and even death have been reported in the literature. Here we present a case wherein the patient had an extraperitoneal bladder perforation that occurred 2 weeks after TUT-BT and immediate postoperative instillation of doxorubicin. Case Presentation A 63-Year-old male, a heavy smoker, with no other co-morbidities, was referred to urology clinic for evaluation and management of a lesion on the left bladder wall that was incidentally detected on a CT scan. He did not have any urinary symptoms on initial presentation. Cystourethroscopy revealed 2 papillary growths on the left lateral bladder wall, which were resected completely and smoothly by a resectoscope using a monopolar energy. Complete resection of the tumor was achieved without any intra-operative complication or perforation. Hemostasis was secured, and a urethral catheter was inserted. In accordance of the EAU guidelines, the patient received an intravesical instillation of 50 ml of doxorubicin diluted in 100 cc of saline 6 hours after the TURBT. During the instillation, his vital signs were stable and he had neither hematuria nor abdominal pain. One hour after the instillation, the catheter was unclamped and was attached to continuous bladder irrigation. Two days after the surgery and in the absence of hematuria, the indwelling catheter was removed, and the patient was discharged home the next day. When seen in the clinic 1 week later the patient was fine with no alarming symptoms. The pathology report showed a low-grade Ta Papillary urothelial carcinoma. Based on the pathological diagnosis and according to the EAU guidelines, adjuvant chemotherapy instillations were planned. Two weeks later, the patient presented with, dysuria, frequency, urgency, as well as pelvic and perineal pain. Physical examination was unremarkable. Urine analysis showed numerous WBC’s, RBC’s, and bacteria. An empirical antibiotics course was commenced. In view of the worsening symptoms and the past history of anal fistula, a contrast enhanced pelvic MRI was performed. It showed a diffusely thickened urinary bladder wall, with evidence of a focal wall defect at its left aspect (the site of the resected tumor). The active urine leak into the perivesical space confirmed the diagnosis of retroperitoneal bladder perforation (Figure 1). Figure. 1. Pelvic MRI (axial view) showing extraperitoneal bladder perforation at the left lateral wall (arrow points to extravasated contrast). The patient was managed conservatively with antibiotics and an indwelling catheter for 3 weeks. A follow-up retrograde CT cystogram showed no improvement and a gross contrast leak from the same site was identified. The catheter was kept for 6 more weeks and the CT cystogram was repeated showing the same results with no evidence of improvement. The patient refused to keep the catheter for a longer period so the option of open surgical repair was discussed with him. Extraperitoneal laparotomy was performed, a perivesical small abscess was drained and cultured, necrotic tissues were debrided, and the bladder wall defect was rimed and closed in 3 layers. Tissue biopsies from the pelvic wall and bladder wall were harvested. A urethral catheter and pelvic drain were kept in place. The postoperative course was uneventful, the drain was removed on the third-day postoperatively, and the patient was discharged home with the urethral catheter in. Histopathological evaluation of the pelvic wall and the bladder biopsies verified acute and chronic inflammation as well as granulation tissue formation. However, both were negative for malignancy. The urinary bladder biopsy also showed extensive foci of dystrophic calcification while the pelvic wall biopsy revealed fat necrosis (Figures 2 and 3). Figure 2. The histologic examination revealed fragments of soft tissue with fat necrosis in the lower left area, mixed acute and chronic inflammatory cell infiltrate and hemorrhage in the upper right area (Hematoxylin & Eosin, 200X). Figure 3. The histologic examination revealed multiple areas of calcifications embedded deep in the tissue with associated crystal-like material suggestive of calcium-oxalate or talc powder crystals (Hematoxylin & Eosin, 200X). A follow up CT cystogram was done 3 weeks after the bladder repair. Although it showed a considerable regression in the size of the bladder defect, interestingly it also showed persistent extra-peritoneal contrast leak from the bladder along it’s left lateral aspect (Figure 4). Figure 4. Retrograde Ct cystogram showing persistent contrast leak from the left lateral aspect after surgical repair. Due to this slow healing, the catheter was kept in. Six weeks later, a follow up CT cystogram showed healing with no extraperitoneal contrast leak (Figure 5). And hence the catheter was removed. Figure 5. Retrograde Ct cystogram showing a healed bladder wall with no contrast leak. Two follow up cystoscopies and bladder biopsy (from an erythematous area on the left lateral wall) with 3 months intervals showed good healing of the bladder wall and no recurrence of the tumor. Discussion Single intravesical instillation with mitomycin C (MMC), epirubicin, or pirarubicin, have shown a beneficial effect in reducing recurrence rate compared to TURBT alone. However, randomized comparisons of individual drugs have not been conducted.3 The high recurrence rate of NMIBC may be attributed to the seeding of floating tumor cells. Immediate chemotherapy instillation has been shown to act by destroying these circulating tumor cells after TURBT. Hence the EAU guidelines recommend initiating the instillation within the first few hours after TURBT.1 Most of the complications of intravesical chemotherapy instillation are minor and can be managed medically. Dysuria and urinary frequency and urgency were the most common complications.4 On the other hand, reporting major complications such as bladder perforation, perirectal abscesses, eosinophilic cystitis, and ureteral stenosis is limited to case reports.5-14 In this case, we described our experience with bladder perforation after immediate intravesical instillation of doxorubicin. One can argue that the perforation has occurred intraoperatively and gone unnoticed. However, in view of the absence of endoscopic evidence of perforation, late presentation of symptoms, and the absence of symptoms during and after the instillation we assume that the Single Dose Intravesical Doxorubicin Instillation caused the perforation rather than complicating an existing one. To explain the mechanism of perforation, Lim et al7 and Cliff et al8 hypothesized that perforation is caused when attenuated muscularis propria gets necrosed after MMC instillation. The presenting complaints of our patient were perineal and pelvic pain as well as urinary irritative symptoms. This is similar to Elmamoun et al9 cohort in which all the 6 patients presented with perineal and pelvic pain. Other symptoms such as fever, urine retention, and recurrent UTI were variably reported by others. Table 1 summarizes the clinical presentation and management of bladder perforation in the previously published case reports. Table1. A summary of the presentation and management of bladder perforation after immediate chemotheraputic agent intravesical instillation. Ref. number The instilled drug Number of patients Initial presentation Management outline Lim et al7 MMC 1 Pelvic and suprapubic pain Exploratry laparotomy and bladder repair after failed conservative management Elmamoun et al9 MMC 6 3 patients presented with pelvic pain Conservative management and urethral catheterization for 2, 26, and 52 weeks Pelvic pain Recurrent UTIs Left ureteric obstruction Partial cystectomy + left to right trans-ureteroureterostomy Pelvic pain, Persistent urinary leak Left ureteric obstruction Augmentation cystoplasty + leftUreteric reimplantationThe patient ended with persistent leak, so a long-term catheter was used Pelvic painSevere LUTS Bilateral ureteric obstruction Urinary diversion (ileal conduit) Penna et al10 MMC 1 Lower abdominal pain and inability to void Analgesia, antibiotics and urethral catheter for 4 weeks Tyritzis et al11 EPI 2 Fever and retropubic pain Conservative: Antibiotics and urethral catheter Fever, lower abdominal pain and cellulitis Exploratry laparotomy and bladder repair after failed conservative management Nieuwenhuijzen et al12 MMC Pelvic pain and fever Exploratry laparotomy and bladder repair after failed conservative management for 1 month Racioppi et al13 MMC Pelvic pain, fever and pancytopenia Exploratry laparotomy and bladder repair after failed conservative management for 3 weeks Hatem and Leifeld14 MMC Pelvic pain Fever and positive blood culture for ecoli as well as purulent discharge from the drain site after the first expolratory laparatomy Conservative management for 10 daysExploratory laparatomy that revealed edematous anterior bladder wall without defectRenewed laparotomy, drainage of the abscess, debridement of the necrotic tissue, mono-J ureteral stents and nephrostomyRadical cystectomy with a urinary diversion through ureterocutaneostomy Shenaq et al15 EPI 3 Strong urge and pain in the lower left abdomen Conservative management with antibiotics and urethral catheterization Abdominal pain and intestinal obstruction Conservative management for 12 daysExploratory laparotomy that revealed infiltrative mass on the top of the bladder causing IO, so colostomy was done.The patient died due to multi organ failure Progressive pain in the lower abdomen Conservative management with antibiotics and urethral catheterization Abbreviations: Ep, epirubicin; MMC, mitomycin C. Concerning the management, almost all the reported cases were initially managed conservatively by urethral catheterization, anticholinergics, and antibiotics. However, this was insufficient to heal the bladder in some cases which needed open repair. Moreover, some ended with cystectomy. In our case, the patient has an extraperitoneal extravasation that failed the conservative management and needed open surgical repair. A striking feature of the healing process that was seen in many cases including ours is the poor and slow healing. In our case, the patient needed 9 weeks for complete healing after proper open 3 layers bladder wall repair.7-9,13,14 While Lim et al attributed the slow healing in their case to the patient’s pre-existing peripheral vascular disease and suboptimal tissue oxygenation, no risk factors were identified in the other cases including our patient who was free of any comorbidities. We suppose that the poor healing is linked to the chemotherapeutic agent inflammatory effect rather than to patient factor. This assumption is backed by the reported characteristic of mitomycin induced skin ulcer in cases of extravasation after intravenous administration. These ulcers are well known for their chronicity and the low tendency of spontaneous healing. Moreover, debridement and soft-tissue reconstruction following extravasation of MMC are recommended for patients with persistent ulceration and pain.15 Another common finding in the cases that mandate open repair was the presence of perivesical soft tissue necrosis and extensive inflammation. Doherty et al observed that more extensive bladder wall and fat necrosis of extravesical tissue in cystectomy specimens occurred when either epirubicin or mitomycin instillation was administered than in that seen following TURBT alone.16 This cytotoxic effect was also reported by Tyritzis et al11 after epirubicin instillation, and by Cliff et al,8 Nieuwenhuijzen et al,12 and Hatem and Leifeld14 after MMC instillation. This cytotoxic effect was recognized herein for the doxorubicin instillation, which is the first time to be reported as a complication of doxorubicin. It is worth to mention that the perforation had no adverse oncological effect on our patient as no tumor recurrence was detected during follow up. Conclusion Immediate intravesical instillations of chemotherapy are not devoid from risk and serious complications as bladder perforation can occur. Whether it’s value in decreasing the recurrence justify the risk of bladder perforation is not our argument in this case report. Nevertheless, this severe complication should be kept in mind, and high index of suspicion will aid in timely management. If perforation is suspected, urethral catheterization and contrasted imaging are mandatory. In the case of failed conservative management surgical repair is warranted even in extraperitoneal leak. Funding:The author(s) received no financial support for the research, authorship, and/or publication of this article. Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Author Contributions: Concept: AA, SAD. Design: MA, SA. Supervision: AA, SAD, MAA. Resources: MA, HM, NB. Materials: AA, MA, NB. Writing manuscript: AA, MA, HM. Critical review: AA, SAD, MAA. Informed Consent: The patient has given his written informed consent to publish his case including publication of images. The study follows the guidelines for human studies and is conducted in accordance with the World Medical Association Declaration of Helsinki. ORCID iD: Hammam Mansi https://orcid.org/0000-0002-4571-4336	DOXORUBICIN	DrugsGivenReaction	CC BY-NC	33473244	18,858,074	2021
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Fat necrosis'.	Delayed Bladder Perforation Related to Immediate Single Dose Intravesical Doxorubicin Instillation After TURBT: A Case Report and Literature Review. Intravesical chemotherapy instillation immediately after tumor resection is a well-known practice in the management of non-muscle invasive bladder cancer. Despite being largely well tolerated in most cases, it is not devoid of severe and life-threatening complications. We present an unusual case of bladder perforation that happened 2 weeks after bladder tumor resection. The patient had received single dose intra-vesical instillation of doxorubicin after TUR-BT. Conservative managements failed to achieve bladder healing; as a result, open surgical repair was performed. To the best of our knowledge, this is the first reported case of bladder perforation after intra-vesical doxorubicin instillation. The occurrence of such a rare serious complication in a mostly safe intervention must be taken into consideration. A high index of suspicion, timely management, and proceeding to more invasive surgical treatments when necessary are cornerstones in the management and preserving the bladder. Introduction Bladder cancer (BC) is the seventh most commonly diagnosed cancer in men worldwide. About 75% of the cases are Non-muscle invasive (NMIBC).1 Transurethral resection of the bladder tumor (TURBT) remains the gold standard of therapy for diagnosing and treating NMIBC. To decrease the recurrence rate of low-risk tumors, a single, immediate, post-operative intravesical instillation of chemotherapy is recommended.1 The most commonly used chemotherapeutic agent is Mitomycin C (MMC), which is an alkylating agent that inhibits DNA synthesis and causes single-strand breakage of DNA and chromosomal breaks.2 Immediate single instillation (SI) is largely well-tolerated. However, severe complications and even death have been reported in the literature. Here we present a case wherein the patient had an extraperitoneal bladder perforation that occurred 2 weeks after TUT-BT and immediate postoperative instillation of doxorubicin. Case Presentation A 63-Year-old male, a heavy smoker, with no other co-morbidities, was referred to urology clinic for evaluation and management of a lesion on the left bladder wall that was incidentally detected on a CT scan. He did not have any urinary symptoms on initial presentation. Cystourethroscopy revealed 2 papillary growths on the left lateral bladder wall, which were resected completely and smoothly by a resectoscope using a monopolar energy. Complete resection of the tumor was achieved without any intra-operative complication or perforation. Hemostasis was secured, and a urethral catheter was inserted. In accordance of the EAU guidelines, the patient received an intravesical instillation of 50 ml of doxorubicin diluted in 100 cc of saline 6 hours after the TURBT. During the instillation, his vital signs were stable and he had neither hematuria nor abdominal pain. One hour after the instillation, the catheter was unclamped and was attached to continuous bladder irrigation. Two days after the surgery and in the absence of hematuria, the indwelling catheter was removed, and the patient was discharged home the next day. When seen in the clinic 1 week later the patient was fine with no alarming symptoms. The pathology report showed a low-grade Ta Papillary urothelial carcinoma. Based on the pathological diagnosis and according to the EAU guidelines, adjuvant chemotherapy instillations were planned. Two weeks later, the patient presented with, dysuria, frequency, urgency, as well as pelvic and perineal pain. Physical examination was unremarkable. Urine analysis showed numerous WBC’s, RBC’s, and bacteria. An empirical antibiotics course was commenced. In view of the worsening symptoms and the past history of anal fistula, a contrast enhanced pelvic MRI was performed. It showed a diffusely thickened urinary bladder wall, with evidence of a focal wall defect at its left aspect (the site of the resected tumor). The active urine leak into the perivesical space confirmed the diagnosis of retroperitoneal bladder perforation (Figure 1). Figure. 1. Pelvic MRI (axial view) showing extraperitoneal bladder perforation at the left lateral wall (arrow points to extravasated contrast). The patient was managed conservatively with antibiotics and an indwelling catheter for 3 weeks. A follow-up retrograde CT cystogram showed no improvement and a gross contrast leak from the same site was identified. The catheter was kept for 6 more weeks and the CT cystogram was repeated showing the same results with no evidence of improvement. The patient refused to keep the catheter for a longer period so the option of open surgical repair was discussed with him. Extraperitoneal laparotomy was performed, a perivesical small abscess was drained and cultured, necrotic tissues were debrided, and the bladder wall defect was rimed and closed in 3 layers. Tissue biopsies from the pelvic wall and bladder wall were harvested. A urethral catheter and pelvic drain were kept in place. The postoperative course was uneventful, the drain was removed on the third-day postoperatively, and the patient was discharged home with the urethral catheter in. Histopathological evaluation of the pelvic wall and the bladder biopsies verified acute and chronic inflammation as well as granulation tissue formation. However, both were negative for malignancy. The urinary bladder biopsy also showed extensive foci of dystrophic calcification while the pelvic wall biopsy revealed fat necrosis (Figures 2 and 3). Figure 2. The histologic examination revealed fragments of soft tissue with fat necrosis in the lower left area, mixed acute and chronic inflammatory cell infiltrate and hemorrhage in the upper right area (Hematoxylin & Eosin, 200X). Figure 3. The histologic examination revealed multiple areas of calcifications embedded deep in the tissue with associated crystal-like material suggestive of calcium-oxalate or talc powder crystals (Hematoxylin & Eosin, 200X). A follow up CT cystogram was done 3 weeks after the bladder repair. Although it showed a considerable regression in the size of the bladder defect, interestingly it also showed persistent extra-peritoneal contrast leak from the bladder along it’s left lateral aspect (Figure 4). Figure 4. Retrograde Ct cystogram showing persistent contrast leak from the left lateral aspect after surgical repair. Due to this slow healing, the catheter was kept in. Six weeks later, a follow up CT cystogram showed healing with no extraperitoneal contrast leak (Figure 5). And hence the catheter was removed. Figure 5. Retrograde Ct cystogram showing a healed bladder wall with no contrast leak. Two follow up cystoscopies and bladder biopsy (from an erythematous area on the left lateral wall) with 3 months intervals showed good healing of the bladder wall and no recurrence of the tumor. Discussion Single intravesical instillation with mitomycin C (MMC), epirubicin, or pirarubicin, have shown a beneficial effect in reducing recurrence rate compared to TURBT alone. However, randomized comparisons of individual drugs have not been conducted.3 The high recurrence rate of NMIBC may be attributed to the seeding of floating tumor cells. Immediate chemotherapy instillation has been shown to act by destroying these circulating tumor cells after TURBT. Hence the EAU guidelines recommend initiating the instillation within the first few hours after TURBT.1 Most of the complications of intravesical chemotherapy instillation are minor and can be managed medically. Dysuria and urinary frequency and urgency were the most common complications.4 On the other hand, reporting major complications such as bladder perforation, perirectal abscesses, eosinophilic cystitis, and ureteral stenosis is limited to case reports.5-14 In this case, we described our experience with bladder perforation after immediate intravesical instillation of doxorubicin. One can argue that the perforation has occurred intraoperatively and gone unnoticed. However, in view of the absence of endoscopic evidence of perforation, late presentation of symptoms, and the absence of symptoms during and after the instillation we assume that the Single Dose Intravesical Doxorubicin Instillation caused the perforation rather than complicating an existing one. To explain the mechanism of perforation, Lim et al7 and Cliff et al8 hypothesized that perforation is caused when attenuated muscularis propria gets necrosed after MMC instillation. The presenting complaints of our patient were perineal and pelvic pain as well as urinary irritative symptoms. This is similar to Elmamoun et al9 cohort in which all the 6 patients presented with perineal and pelvic pain. Other symptoms such as fever, urine retention, and recurrent UTI were variably reported by others. Table 1 summarizes the clinical presentation and management of bladder perforation in the previously published case reports. Table1. A summary of the presentation and management of bladder perforation after immediate chemotheraputic agent intravesical instillation. Ref. number The instilled drug Number of patients Initial presentation Management outline Lim et al7 MMC 1 Pelvic and suprapubic pain Exploratry laparotomy and bladder repair after failed conservative management Elmamoun et al9 MMC 6 3 patients presented with pelvic pain Conservative management and urethral catheterization for 2, 26, and 52 weeks Pelvic pain Recurrent UTIs Left ureteric obstruction Partial cystectomy + left to right trans-ureteroureterostomy Pelvic pain, Persistent urinary leak Left ureteric obstruction Augmentation cystoplasty + leftUreteric reimplantationThe patient ended with persistent leak, so a long-term catheter was used Pelvic painSevere LUTS Bilateral ureteric obstruction Urinary diversion (ileal conduit) Penna et al10 MMC 1 Lower abdominal pain and inability to void Analgesia, antibiotics and urethral catheter for 4 weeks Tyritzis et al11 EPI 2 Fever and retropubic pain Conservative: Antibiotics and urethral catheter Fever, lower abdominal pain and cellulitis Exploratry laparotomy and bladder repair after failed conservative management Nieuwenhuijzen et al12 MMC Pelvic pain and fever Exploratry laparotomy and bladder repair after failed conservative management for 1 month Racioppi et al13 MMC Pelvic pain, fever and pancytopenia Exploratry laparotomy and bladder repair after failed conservative management for 3 weeks Hatem and Leifeld14 MMC Pelvic pain Fever and positive blood culture for ecoli as well as purulent discharge from the drain site after the first expolratory laparatomy Conservative management for 10 daysExploratory laparatomy that revealed edematous anterior bladder wall without defectRenewed laparotomy, drainage of the abscess, debridement of the necrotic tissue, mono-J ureteral stents and nephrostomyRadical cystectomy with a urinary diversion through ureterocutaneostomy Shenaq et al15 EPI 3 Strong urge and pain in the lower left abdomen Conservative management with antibiotics and urethral catheterization Abdominal pain and intestinal obstruction Conservative management for 12 daysExploratory laparotomy that revealed infiltrative mass on the top of the bladder causing IO, so colostomy was done.The patient died due to multi organ failure Progressive pain in the lower abdomen Conservative management with antibiotics and urethral catheterization Abbreviations: Ep, epirubicin; MMC, mitomycin C. Concerning the management, almost all the reported cases were initially managed conservatively by urethral catheterization, anticholinergics, and antibiotics. However, this was insufficient to heal the bladder in some cases which needed open repair. Moreover, some ended with cystectomy. In our case, the patient has an extraperitoneal extravasation that failed the conservative management and needed open surgical repair. A striking feature of the healing process that was seen in many cases including ours is the poor and slow healing. In our case, the patient needed 9 weeks for complete healing after proper open 3 layers bladder wall repair.7-9,13,14 While Lim et al attributed the slow healing in their case to the patient’s pre-existing peripheral vascular disease and suboptimal tissue oxygenation, no risk factors were identified in the other cases including our patient who was free of any comorbidities. We suppose that the poor healing is linked to the chemotherapeutic agent inflammatory effect rather than to patient factor. This assumption is backed by the reported characteristic of mitomycin induced skin ulcer in cases of extravasation after intravenous administration. These ulcers are well known for their chronicity and the low tendency of spontaneous healing. Moreover, debridement and soft-tissue reconstruction following extravasation of MMC are recommended for patients with persistent ulceration and pain.15 Another common finding in the cases that mandate open repair was the presence of perivesical soft tissue necrosis and extensive inflammation. Doherty et al observed that more extensive bladder wall and fat necrosis of extravesical tissue in cystectomy specimens occurred when either epirubicin or mitomycin instillation was administered than in that seen following TURBT alone.16 This cytotoxic effect was also reported by Tyritzis et al11 after epirubicin instillation, and by Cliff et al,8 Nieuwenhuijzen et al,12 and Hatem and Leifeld14 after MMC instillation. This cytotoxic effect was recognized herein for the doxorubicin instillation, which is the first time to be reported as a complication of doxorubicin. It is worth to mention that the perforation had no adverse oncological effect on our patient as no tumor recurrence was detected during follow up. Conclusion Immediate intravesical instillations of chemotherapy are not devoid from risk and serious complications as bladder perforation can occur. Whether it’s value in decreasing the recurrence justify the risk of bladder perforation is not our argument in this case report. Nevertheless, this severe complication should be kept in mind, and high index of suspicion will aid in timely management. If perforation is suspected, urethral catheterization and contrasted imaging are mandatory. In the case of failed conservative management surgical repair is warranted even in extraperitoneal leak. Funding:The author(s) received no financial support for the research, authorship, and/or publication of this article. Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Author Contributions: Concept: AA, SAD. Design: MA, SA. Supervision: AA, SAD, MAA. Resources: MA, HM, NB. Materials: AA, MA, NB. Writing manuscript: AA, MA, HM. Critical review: AA, SAD, MAA. Informed Consent: The patient has given his written informed consent to publish his case including publication of images. The study follows the guidelines for human studies and is conducted in accordance with the World Medical Association Declaration of Helsinki. ORCID iD: Hammam Mansi https://orcid.org/0000-0002-4571-4336	DOXORUBICIN	DrugsGivenReaction	CC BY-NC	33473244	18,858,074	2021
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Impaired healing'.	Delayed Bladder Perforation Related to Immediate Single Dose Intravesical Doxorubicin Instillation After TURBT: A Case Report and Literature Review. Intravesical chemotherapy instillation immediately after tumor resection is a well-known practice in the management of non-muscle invasive bladder cancer. Despite being largely well tolerated in most cases, it is not devoid of severe and life-threatening complications. We present an unusual case of bladder perforation that happened 2 weeks after bladder tumor resection. The patient had received single dose intra-vesical instillation of doxorubicin after TUR-BT. Conservative managements failed to achieve bladder healing; as a result, open surgical repair was performed. To the best of our knowledge, this is the first reported case of bladder perforation after intra-vesical doxorubicin instillation. The occurrence of such a rare serious complication in a mostly safe intervention must be taken into consideration. A high index of suspicion, timely management, and proceeding to more invasive surgical treatments when necessary are cornerstones in the management and preserving the bladder. Introduction Bladder cancer (BC) is the seventh most commonly diagnosed cancer in men worldwide. About 75% of the cases are Non-muscle invasive (NMIBC).1 Transurethral resection of the bladder tumor (TURBT) remains the gold standard of therapy for diagnosing and treating NMIBC. To decrease the recurrence rate of low-risk tumors, a single, immediate, post-operative intravesical instillation of chemotherapy is recommended.1 The most commonly used chemotherapeutic agent is Mitomycin C (MMC), which is an alkylating agent that inhibits DNA synthesis and causes single-strand breakage of DNA and chromosomal breaks.2 Immediate single instillation (SI) is largely well-tolerated. However, severe complications and even death have been reported in the literature. Here we present a case wherein the patient had an extraperitoneal bladder perforation that occurred 2 weeks after TUT-BT and immediate postoperative instillation of doxorubicin. Case Presentation A 63-Year-old male, a heavy smoker, with no other co-morbidities, was referred to urology clinic for evaluation and management of a lesion on the left bladder wall that was incidentally detected on a CT scan. He did not have any urinary symptoms on initial presentation. Cystourethroscopy revealed 2 papillary growths on the left lateral bladder wall, which were resected completely and smoothly by a resectoscope using a monopolar energy. Complete resection of the tumor was achieved without any intra-operative complication or perforation. Hemostasis was secured, and a urethral catheter was inserted. In accordance of the EAU guidelines, the patient received an intravesical instillation of 50 ml of doxorubicin diluted in 100 cc of saline 6 hours after the TURBT. During the instillation, his vital signs were stable and he had neither hematuria nor abdominal pain. One hour after the instillation, the catheter was unclamped and was attached to continuous bladder irrigation. Two days after the surgery and in the absence of hematuria, the indwelling catheter was removed, and the patient was discharged home the next day. When seen in the clinic 1 week later the patient was fine with no alarming symptoms. The pathology report showed a low-grade Ta Papillary urothelial carcinoma. Based on the pathological diagnosis and according to the EAU guidelines, adjuvant chemotherapy instillations were planned. Two weeks later, the patient presented with, dysuria, frequency, urgency, as well as pelvic and perineal pain. Physical examination was unremarkable. Urine analysis showed numerous WBC’s, RBC’s, and bacteria. An empirical antibiotics course was commenced. In view of the worsening symptoms and the past history of anal fistula, a contrast enhanced pelvic MRI was performed. It showed a diffusely thickened urinary bladder wall, with evidence of a focal wall defect at its left aspect (the site of the resected tumor). The active urine leak into the perivesical space confirmed the diagnosis of retroperitoneal bladder perforation (Figure 1). Figure. 1. Pelvic MRI (axial view) showing extraperitoneal bladder perforation at the left lateral wall (arrow points to extravasated contrast). The patient was managed conservatively with antibiotics and an indwelling catheter for 3 weeks. A follow-up retrograde CT cystogram showed no improvement and a gross contrast leak from the same site was identified. The catheter was kept for 6 more weeks and the CT cystogram was repeated showing the same results with no evidence of improvement. The patient refused to keep the catheter for a longer period so the option of open surgical repair was discussed with him. Extraperitoneal laparotomy was performed, a perivesical small abscess was drained and cultured, necrotic tissues were debrided, and the bladder wall defect was rimed and closed in 3 layers. Tissue biopsies from the pelvic wall and bladder wall were harvested. A urethral catheter and pelvic drain were kept in place. The postoperative course was uneventful, the drain was removed on the third-day postoperatively, and the patient was discharged home with the urethral catheter in. Histopathological evaluation of the pelvic wall and the bladder biopsies verified acute and chronic inflammation as well as granulation tissue formation. However, both were negative for malignancy. The urinary bladder biopsy also showed extensive foci of dystrophic calcification while the pelvic wall biopsy revealed fat necrosis (Figures 2 and 3). Figure 2. The histologic examination revealed fragments of soft tissue with fat necrosis in the lower left area, mixed acute and chronic inflammatory cell infiltrate and hemorrhage in the upper right area (Hematoxylin & Eosin, 200X). Figure 3. The histologic examination revealed multiple areas of calcifications embedded deep in the tissue with associated crystal-like material suggestive of calcium-oxalate or talc powder crystals (Hematoxylin & Eosin, 200X). A follow up CT cystogram was done 3 weeks after the bladder repair. Although it showed a considerable regression in the size of the bladder defect, interestingly it also showed persistent extra-peritoneal contrast leak from the bladder along it’s left lateral aspect (Figure 4). Figure 4. Retrograde Ct cystogram showing persistent contrast leak from the left lateral aspect after surgical repair. Due to this slow healing, the catheter was kept in. Six weeks later, a follow up CT cystogram showed healing with no extraperitoneal contrast leak (Figure 5). And hence the catheter was removed. Figure 5. Retrograde Ct cystogram showing a healed bladder wall with no contrast leak. Two follow up cystoscopies and bladder biopsy (from an erythematous area on the left lateral wall) with 3 months intervals showed good healing of the bladder wall and no recurrence of the tumor. Discussion Single intravesical instillation with mitomycin C (MMC), epirubicin, or pirarubicin, have shown a beneficial effect in reducing recurrence rate compared to TURBT alone. However, randomized comparisons of individual drugs have not been conducted.3 The high recurrence rate of NMIBC may be attributed to the seeding of floating tumor cells. Immediate chemotherapy instillation has been shown to act by destroying these circulating tumor cells after TURBT. Hence the EAU guidelines recommend initiating the instillation within the first few hours after TURBT.1 Most of the complications of intravesical chemotherapy instillation are minor and can be managed medically. Dysuria and urinary frequency and urgency were the most common complications.4 On the other hand, reporting major complications such as bladder perforation, perirectal abscesses, eosinophilic cystitis, and ureteral stenosis is limited to case reports.5-14 In this case, we described our experience with bladder perforation after immediate intravesical instillation of doxorubicin. One can argue that the perforation has occurred intraoperatively and gone unnoticed. However, in view of the absence of endoscopic evidence of perforation, late presentation of symptoms, and the absence of symptoms during and after the instillation we assume that the Single Dose Intravesical Doxorubicin Instillation caused the perforation rather than complicating an existing one. To explain the mechanism of perforation, Lim et al7 and Cliff et al8 hypothesized that perforation is caused when attenuated muscularis propria gets necrosed after MMC instillation. The presenting complaints of our patient were perineal and pelvic pain as well as urinary irritative symptoms. This is similar to Elmamoun et al9 cohort in which all the 6 patients presented with perineal and pelvic pain. Other symptoms such as fever, urine retention, and recurrent UTI were variably reported by others. Table 1 summarizes the clinical presentation and management of bladder perforation in the previously published case reports. Table1. A summary of the presentation and management of bladder perforation after immediate chemotheraputic agent intravesical instillation. Ref. number The instilled drug Number of patients Initial presentation Management outline Lim et al7 MMC 1 Pelvic and suprapubic pain Exploratry laparotomy and bladder repair after failed conservative management Elmamoun et al9 MMC 6 3 patients presented with pelvic pain Conservative management and urethral catheterization for 2, 26, and 52 weeks Pelvic pain Recurrent UTIs Left ureteric obstruction Partial cystectomy + left to right trans-ureteroureterostomy Pelvic pain, Persistent urinary leak Left ureteric obstruction Augmentation cystoplasty + leftUreteric reimplantationThe patient ended with persistent leak, so a long-term catheter was used Pelvic painSevere LUTS Bilateral ureteric obstruction Urinary diversion (ileal conduit) Penna et al10 MMC 1 Lower abdominal pain and inability to void Analgesia, antibiotics and urethral catheter for 4 weeks Tyritzis et al11 EPI 2 Fever and retropubic pain Conservative: Antibiotics and urethral catheter Fever, lower abdominal pain and cellulitis Exploratry laparotomy and bladder repair after failed conservative management Nieuwenhuijzen et al12 MMC Pelvic pain and fever Exploratry laparotomy and bladder repair after failed conservative management for 1 month Racioppi et al13 MMC Pelvic pain, fever and pancytopenia Exploratry laparotomy and bladder repair after failed conservative management for 3 weeks Hatem and Leifeld14 MMC Pelvic pain Fever and positive blood culture for ecoli as well as purulent discharge from the drain site after the first expolratory laparatomy Conservative management for 10 daysExploratory laparatomy that revealed edematous anterior bladder wall without defectRenewed laparotomy, drainage of the abscess, debridement of the necrotic tissue, mono-J ureteral stents and nephrostomyRadical cystectomy with a urinary diversion through ureterocutaneostomy Shenaq et al15 EPI 3 Strong urge and pain in the lower left abdomen Conservative management with antibiotics and urethral catheterization Abdominal pain and intestinal obstruction Conservative management for 12 daysExploratory laparotomy that revealed infiltrative mass on the top of the bladder causing IO, so colostomy was done.The patient died due to multi organ failure Progressive pain in the lower abdomen Conservative management with antibiotics and urethral catheterization Abbreviations: Ep, epirubicin; MMC, mitomycin C. Concerning the management, almost all the reported cases were initially managed conservatively by urethral catheterization, anticholinergics, and antibiotics. However, this was insufficient to heal the bladder in some cases which needed open repair. Moreover, some ended with cystectomy. In our case, the patient has an extraperitoneal extravasation that failed the conservative management and needed open surgical repair. A striking feature of the healing process that was seen in many cases including ours is the poor and slow healing. In our case, the patient needed 9 weeks for complete healing after proper open 3 layers bladder wall repair.7-9,13,14 While Lim et al attributed the slow healing in their case to the patient’s pre-existing peripheral vascular disease and suboptimal tissue oxygenation, no risk factors were identified in the other cases including our patient who was free of any comorbidities. We suppose that the poor healing is linked to the chemotherapeutic agent inflammatory effect rather than to patient factor. This assumption is backed by the reported characteristic of mitomycin induced skin ulcer in cases of extravasation after intravenous administration. These ulcers are well known for their chronicity and the low tendency of spontaneous healing. Moreover, debridement and soft-tissue reconstruction following extravasation of MMC are recommended for patients with persistent ulceration and pain.15 Another common finding in the cases that mandate open repair was the presence of perivesical soft tissue necrosis and extensive inflammation. Doherty et al observed that more extensive bladder wall and fat necrosis of extravesical tissue in cystectomy specimens occurred when either epirubicin or mitomycin instillation was administered than in that seen following TURBT alone.16 This cytotoxic effect was also reported by Tyritzis et al11 after epirubicin instillation, and by Cliff et al,8 Nieuwenhuijzen et al,12 and Hatem and Leifeld14 after MMC instillation. This cytotoxic effect was recognized herein for the doxorubicin instillation, which is the first time to be reported as a complication of doxorubicin. It is worth to mention that the perforation had no adverse oncological effect on our patient as no tumor recurrence was detected during follow up. Conclusion Immediate intravesical instillations of chemotherapy are not devoid from risk and serious complications as bladder perforation can occur. Whether it’s value in decreasing the recurrence justify the risk of bladder perforation is not our argument in this case report. Nevertheless, this severe complication should be kept in mind, and high index of suspicion will aid in timely management. If perforation is suspected, urethral catheterization and contrasted imaging are mandatory. In the case of failed conservative management surgical repair is warranted even in extraperitoneal leak. Funding:The author(s) received no financial support for the research, authorship, and/or publication of this article. Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Author Contributions: Concept: AA, SAD. Design: MA, SA. Supervision: AA, SAD, MAA. Resources: MA, HM, NB. Materials: AA, MA, NB. Writing manuscript: AA, MA, HM. Critical review: AA, SAD, MAA. Informed Consent: The patient has given his written informed consent to publish his case including publication of images. The study follows the guidelines for human studies and is conducted in accordance with the World Medical Association Declaration of Helsinki. ORCID iD: Hammam Mansi https://orcid.org/0000-0002-4571-4336	DOXORUBICIN	DrugsGivenReaction	CC BY-NC	33473244	18,858,074	2021
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Micturition urgency'.	Delayed Bladder Perforation Related to Immediate Single Dose Intravesical Doxorubicin Instillation After TURBT: A Case Report and Literature Review. Intravesical chemotherapy instillation immediately after tumor resection is a well-known practice in the management of non-muscle invasive bladder cancer. Despite being largely well tolerated in most cases, it is not devoid of severe and life-threatening complications. We present an unusual case of bladder perforation that happened 2 weeks after bladder tumor resection. The patient had received single dose intra-vesical instillation of doxorubicin after TUR-BT. Conservative managements failed to achieve bladder healing; as a result, open surgical repair was performed. To the best of our knowledge, this is the first reported case of bladder perforation after intra-vesical doxorubicin instillation. The occurrence of such a rare serious complication in a mostly safe intervention must be taken into consideration. A high index of suspicion, timely management, and proceeding to more invasive surgical treatments when necessary are cornerstones in the management and preserving the bladder. Introduction Bladder cancer (BC) is the seventh most commonly diagnosed cancer in men worldwide. About 75% of the cases are Non-muscle invasive (NMIBC).1 Transurethral resection of the bladder tumor (TURBT) remains the gold standard of therapy for diagnosing and treating NMIBC. To decrease the recurrence rate of low-risk tumors, a single, immediate, post-operative intravesical instillation of chemotherapy is recommended.1 The most commonly used chemotherapeutic agent is Mitomycin C (MMC), which is an alkylating agent that inhibits DNA synthesis and causes single-strand breakage of DNA and chromosomal breaks.2 Immediate single instillation (SI) is largely well-tolerated. However, severe complications and even death have been reported in the literature. Here we present a case wherein the patient had an extraperitoneal bladder perforation that occurred 2 weeks after TUT-BT and immediate postoperative instillation of doxorubicin. Case Presentation A 63-Year-old male, a heavy smoker, with no other co-morbidities, was referred to urology clinic for evaluation and management of a lesion on the left bladder wall that was incidentally detected on a CT scan. He did not have any urinary symptoms on initial presentation. Cystourethroscopy revealed 2 papillary growths on the left lateral bladder wall, which were resected completely and smoothly by a resectoscope using a monopolar energy. Complete resection of the tumor was achieved without any intra-operative complication or perforation. Hemostasis was secured, and a urethral catheter was inserted. In accordance of the EAU guidelines, the patient received an intravesical instillation of 50 ml of doxorubicin diluted in 100 cc of saline 6 hours after the TURBT. During the instillation, his vital signs were stable and he had neither hematuria nor abdominal pain. One hour after the instillation, the catheter was unclamped and was attached to continuous bladder irrigation. Two days after the surgery and in the absence of hematuria, the indwelling catheter was removed, and the patient was discharged home the next day. When seen in the clinic 1 week later the patient was fine with no alarming symptoms. The pathology report showed a low-grade Ta Papillary urothelial carcinoma. Based on the pathological diagnosis and according to the EAU guidelines, adjuvant chemotherapy instillations were planned. Two weeks later, the patient presented with, dysuria, frequency, urgency, as well as pelvic and perineal pain. Physical examination was unremarkable. Urine analysis showed numerous WBC’s, RBC’s, and bacteria. An empirical antibiotics course was commenced. In view of the worsening symptoms and the past history of anal fistula, a contrast enhanced pelvic MRI was performed. It showed a diffusely thickened urinary bladder wall, with evidence of a focal wall defect at its left aspect (the site of the resected tumor). The active urine leak into the perivesical space confirmed the diagnosis of retroperitoneal bladder perforation (Figure 1). Figure. 1. Pelvic MRI (axial view) showing extraperitoneal bladder perforation at the left lateral wall (arrow points to extravasated contrast). The patient was managed conservatively with antibiotics and an indwelling catheter for 3 weeks. A follow-up retrograde CT cystogram showed no improvement and a gross contrast leak from the same site was identified. The catheter was kept for 6 more weeks and the CT cystogram was repeated showing the same results with no evidence of improvement. The patient refused to keep the catheter for a longer period so the option of open surgical repair was discussed with him. Extraperitoneal laparotomy was performed, a perivesical small abscess was drained and cultured, necrotic tissues were debrided, and the bladder wall defect was rimed and closed in 3 layers. Tissue biopsies from the pelvic wall and bladder wall were harvested. A urethral catheter and pelvic drain were kept in place. The postoperative course was uneventful, the drain was removed on the third-day postoperatively, and the patient was discharged home with the urethral catheter in. Histopathological evaluation of the pelvic wall and the bladder biopsies verified acute and chronic inflammation as well as granulation tissue formation. However, both were negative for malignancy. The urinary bladder biopsy also showed extensive foci of dystrophic calcification while the pelvic wall biopsy revealed fat necrosis (Figures 2 and 3). Figure 2. The histologic examination revealed fragments of soft tissue with fat necrosis in the lower left area, mixed acute and chronic inflammatory cell infiltrate and hemorrhage in the upper right area (Hematoxylin & Eosin, 200X). Figure 3. The histologic examination revealed multiple areas of calcifications embedded deep in the tissue with associated crystal-like material suggestive of calcium-oxalate or talc powder crystals (Hematoxylin & Eosin, 200X). A follow up CT cystogram was done 3 weeks after the bladder repair. Although it showed a considerable regression in the size of the bladder defect, interestingly it also showed persistent extra-peritoneal contrast leak from the bladder along it’s left lateral aspect (Figure 4). Figure 4. Retrograde Ct cystogram showing persistent contrast leak from the left lateral aspect after surgical repair. Due to this slow healing, the catheter was kept in. Six weeks later, a follow up CT cystogram showed healing with no extraperitoneal contrast leak (Figure 5). And hence the catheter was removed. Figure 5. Retrograde Ct cystogram showing a healed bladder wall with no contrast leak. Two follow up cystoscopies and bladder biopsy (from an erythematous area on the left lateral wall) with 3 months intervals showed good healing of the bladder wall and no recurrence of the tumor. Discussion Single intravesical instillation with mitomycin C (MMC), epirubicin, or pirarubicin, have shown a beneficial effect in reducing recurrence rate compared to TURBT alone. However, randomized comparisons of individual drugs have not been conducted.3 The high recurrence rate of NMIBC may be attributed to the seeding of floating tumor cells. Immediate chemotherapy instillation has been shown to act by destroying these circulating tumor cells after TURBT. Hence the EAU guidelines recommend initiating the instillation within the first few hours after TURBT.1 Most of the complications of intravesical chemotherapy instillation are minor and can be managed medically. Dysuria and urinary frequency and urgency were the most common complications.4 On the other hand, reporting major complications such as bladder perforation, perirectal abscesses, eosinophilic cystitis, and ureteral stenosis is limited to case reports.5-14 In this case, we described our experience with bladder perforation after immediate intravesical instillation of doxorubicin. One can argue that the perforation has occurred intraoperatively and gone unnoticed. However, in view of the absence of endoscopic evidence of perforation, late presentation of symptoms, and the absence of symptoms during and after the instillation we assume that the Single Dose Intravesical Doxorubicin Instillation caused the perforation rather than complicating an existing one. To explain the mechanism of perforation, Lim et al7 and Cliff et al8 hypothesized that perforation is caused when attenuated muscularis propria gets necrosed after MMC instillation. The presenting complaints of our patient were perineal and pelvic pain as well as urinary irritative symptoms. This is similar to Elmamoun et al9 cohort in which all the 6 patients presented with perineal and pelvic pain. Other symptoms such as fever, urine retention, and recurrent UTI were variably reported by others. Table 1 summarizes the clinical presentation and management of bladder perforation in the previously published case reports. Table1. A summary of the presentation and management of bladder perforation after immediate chemotheraputic agent intravesical instillation. Ref. number The instilled drug Number of patients Initial presentation Management outline Lim et al7 MMC 1 Pelvic and suprapubic pain Exploratry laparotomy and bladder repair after failed conservative management Elmamoun et al9 MMC 6 3 patients presented with pelvic pain Conservative management and urethral catheterization for 2, 26, and 52 weeks Pelvic pain Recurrent UTIs Left ureteric obstruction Partial cystectomy + left to right trans-ureteroureterostomy Pelvic pain, Persistent urinary leak Left ureteric obstruction Augmentation cystoplasty + leftUreteric reimplantationThe patient ended with persistent leak, so a long-term catheter was used Pelvic painSevere LUTS Bilateral ureteric obstruction Urinary diversion (ileal conduit) Penna et al10 MMC 1 Lower abdominal pain and inability to void Analgesia, antibiotics and urethral catheter for 4 weeks Tyritzis et al11 EPI 2 Fever and retropubic pain Conservative: Antibiotics and urethral catheter Fever, lower abdominal pain and cellulitis Exploratry laparotomy and bladder repair after failed conservative management Nieuwenhuijzen et al12 MMC Pelvic pain and fever Exploratry laparotomy and bladder repair after failed conservative management for 1 month Racioppi et al13 MMC Pelvic pain, fever and pancytopenia Exploratry laparotomy and bladder repair after failed conservative management for 3 weeks Hatem and Leifeld14 MMC Pelvic pain Fever and positive blood culture for ecoli as well as purulent discharge from the drain site after the first expolratory laparatomy Conservative management for 10 daysExploratory laparatomy that revealed edematous anterior bladder wall without defectRenewed laparotomy, drainage of the abscess, debridement of the necrotic tissue, mono-J ureteral stents and nephrostomyRadical cystectomy with a urinary diversion through ureterocutaneostomy Shenaq et al15 EPI 3 Strong urge and pain in the lower left abdomen Conservative management with antibiotics and urethral catheterization Abdominal pain and intestinal obstruction Conservative management for 12 daysExploratory laparotomy that revealed infiltrative mass on the top of the bladder causing IO, so colostomy was done.The patient died due to multi organ failure Progressive pain in the lower abdomen Conservative management with antibiotics and urethral catheterization Abbreviations: Ep, epirubicin; MMC, mitomycin C. Concerning the management, almost all the reported cases were initially managed conservatively by urethral catheterization, anticholinergics, and antibiotics. However, this was insufficient to heal the bladder in some cases which needed open repair. Moreover, some ended with cystectomy. In our case, the patient has an extraperitoneal extravasation that failed the conservative management and needed open surgical repair. A striking feature of the healing process that was seen in many cases including ours is the poor and slow healing. In our case, the patient needed 9 weeks for complete healing after proper open 3 layers bladder wall repair.7-9,13,14 While Lim et al attributed the slow healing in their case to the patient’s pre-existing peripheral vascular disease and suboptimal tissue oxygenation, no risk factors were identified in the other cases including our patient who was free of any comorbidities. We suppose that the poor healing is linked to the chemotherapeutic agent inflammatory effect rather than to patient factor. This assumption is backed by the reported characteristic of mitomycin induced skin ulcer in cases of extravasation after intravenous administration. These ulcers are well known for their chronicity and the low tendency of spontaneous healing. Moreover, debridement and soft-tissue reconstruction following extravasation of MMC are recommended for patients with persistent ulceration and pain.15 Another common finding in the cases that mandate open repair was the presence of perivesical soft tissue necrosis and extensive inflammation. Doherty et al observed that more extensive bladder wall and fat necrosis of extravesical tissue in cystectomy specimens occurred when either epirubicin or mitomycin instillation was administered than in that seen following TURBT alone.16 This cytotoxic effect was also reported by Tyritzis et al11 after epirubicin instillation, and by Cliff et al,8 Nieuwenhuijzen et al,12 and Hatem and Leifeld14 after MMC instillation. This cytotoxic effect was recognized herein for the doxorubicin instillation, which is the first time to be reported as a complication of doxorubicin. It is worth to mention that the perforation had no adverse oncological effect on our patient as no tumor recurrence was detected during follow up. Conclusion Immediate intravesical instillations of chemotherapy are not devoid from risk and serious complications as bladder perforation can occur. Whether it’s value in decreasing the recurrence justify the risk of bladder perforation is not our argument in this case report. Nevertheless, this severe complication should be kept in mind, and high index of suspicion will aid in timely management. If perforation is suspected, urethral catheterization and contrasted imaging are mandatory. In the case of failed conservative management surgical repair is warranted even in extraperitoneal leak. Funding:The author(s) received no financial support for the research, authorship, and/or publication of this article. Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Author Contributions: Concept: AA, SAD. Design: MA, SA. Supervision: AA, SAD, MAA. Resources: MA, HM, NB. Materials: AA, MA, NB. Writing manuscript: AA, MA, HM. Critical review: AA, SAD, MAA. Informed Consent: The patient has given his written informed consent to publish his case including publication of images. The study follows the guidelines for human studies and is conducted in accordance with the World Medical Association Declaration of Helsinki. ORCID iD: Hammam Mansi https://orcid.org/0000-0002-4571-4336	DOXORUBICIN	DrugsGivenReaction	CC BY-NC	33473244	18,858,074	2021
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Off label use'.	Delayed Bladder Perforation Related to Immediate Single Dose Intravesical Doxorubicin Instillation After TURBT: A Case Report and Literature Review. Intravesical chemotherapy instillation immediately after tumor resection is a well-known practice in the management of non-muscle invasive bladder cancer. Despite being largely well tolerated in most cases, it is not devoid of severe and life-threatening complications. We present an unusual case of bladder perforation that happened 2 weeks after bladder tumor resection. The patient had received single dose intra-vesical instillation of doxorubicin after TUR-BT. Conservative managements failed to achieve bladder healing; as a result, open surgical repair was performed. To the best of our knowledge, this is the first reported case of bladder perforation after intra-vesical doxorubicin instillation. The occurrence of such a rare serious complication in a mostly safe intervention must be taken into consideration. A high index of suspicion, timely management, and proceeding to more invasive surgical treatments when necessary are cornerstones in the management and preserving the bladder. Introduction Bladder cancer (BC) is the seventh most commonly diagnosed cancer in men worldwide. About 75% of the cases are Non-muscle invasive (NMIBC).1 Transurethral resection of the bladder tumor (TURBT) remains the gold standard of therapy for diagnosing and treating NMIBC. To decrease the recurrence rate of low-risk tumors, a single, immediate, post-operative intravesical instillation of chemotherapy is recommended.1 The most commonly used chemotherapeutic agent is Mitomycin C (MMC), which is an alkylating agent that inhibits DNA synthesis and causes single-strand breakage of DNA and chromosomal breaks.2 Immediate single instillation (SI) is largely well-tolerated. However, severe complications and even death have been reported in the literature. Here we present a case wherein the patient had an extraperitoneal bladder perforation that occurred 2 weeks after TUT-BT and immediate postoperative instillation of doxorubicin. Case Presentation A 63-Year-old male, a heavy smoker, with no other co-morbidities, was referred to urology clinic for evaluation and management of a lesion on the left bladder wall that was incidentally detected on a CT scan. He did not have any urinary symptoms on initial presentation. Cystourethroscopy revealed 2 papillary growths on the left lateral bladder wall, which were resected completely and smoothly by a resectoscope using a monopolar energy. Complete resection of the tumor was achieved without any intra-operative complication or perforation. Hemostasis was secured, and a urethral catheter was inserted. In accordance of the EAU guidelines, the patient received an intravesical instillation of 50 ml of doxorubicin diluted in 100 cc of saline 6 hours after the TURBT. During the instillation, his vital signs were stable and he had neither hematuria nor abdominal pain. One hour after the instillation, the catheter was unclamped and was attached to continuous bladder irrigation. Two days after the surgery and in the absence of hematuria, the indwelling catheter was removed, and the patient was discharged home the next day. When seen in the clinic 1 week later the patient was fine with no alarming symptoms. The pathology report showed a low-grade Ta Papillary urothelial carcinoma. Based on the pathological diagnosis and according to the EAU guidelines, adjuvant chemotherapy instillations were planned. Two weeks later, the patient presented with, dysuria, frequency, urgency, as well as pelvic and perineal pain. Physical examination was unremarkable. Urine analysis showed numerous WBC’s, RBC’s, and bacteria. An empirical antibiotics course was commenced. In view of the worsening symptoms and the past history of anal fistula, a contrast enhanced pelvic MRI was performed. It showed a diffusely thickened urinary bladder wall, with evidence of a focal wall defect at its left aspect (the site of the resected tumor). The active urine leak into the perivesical space confirmed the diagnosis of retroperitoneal bladder perforation (Figure 1). Figure. 1. Pelvic MRI (axial view) showing extraperitoneal bladder perforation at the left lateral wall (arrow points to extravasated contrast). The patient was managed conservatively with antibiotics and an indwelling catheter for 3 weeks. A follow-up retrograde CT cystogram showed no improvement and a gross contrast leak from the same site was identified. The catheter was kept for 6 more weeks and the CT cystogram was repeated showing the same results with no evidence of improvement. The patient refused to keep the catheter for a longer period so the option of open surgical repair was discussed with him. Extraperitoneal laparotomy was performed, a perivesical small abscess was drained and cultured, necrotic tissues were debrided, and the bladder wall defect was rimed and closed in 3 layers. Tissue biopsies from the pelvic wall and bladder wall were harvested. A urethral catheter and pelvic drain were kept in place. The postoperative course was uneventful, the drain was removed on the third-day postoperatively, and the patient was discharged home with the urethral catheter in. Histopathological evaluation of the pelvic wall and the bladder biopsies verified acute and chronic inflammation as well as granulation tissue formation. However, both were negative for malignancy. The urinary bladder biopsy also showed extensive foci of dystrophic calcification while the pelvic wall biopsy revealed fat necrosis (Figures 2 and 3). Figure 2. The histologic examination revealed fragments of soft tissue with fat necrosis in the lower left area, mixed acute and chronic inflammatory cell infiltrate and hemorrhage in the upper right area (Hematoxylin & Eosin, 200X). Figure 3. The histologic examination revealed multiple areas of calcifications embedded deep in the tissue with associated crystal-like material suggestive of calcium-oxalate or talc powder crystals (Hematoxylin & Eosin, 200X). A follow up CT cystogram was done 3 weeks after the bladder repair. Although it showed a considerable regression in the size of the bladder defect, interestingly it also showed persistent extra-peritoneal contrast leak from the bladder along it’s left lateral aspect (Figure 4). Figure 4. Retrograde Ct cystogram showing persistent contrast leak from the left lateral aspect after surgical repair. Due to this slow healing, the catheter was kept in. Six weeks later, a follow up CT cystogram showed healing with no extraperitoneal contrast leak (Figure 5). And hence the catheter was removed. Figure 5. Retrograde Ct cystogram showing a healed bladder wall with no contrast leak. Two follow up cystoscopies and bladder biopsy (from an erythematous area on the left lateral wall) with 3 months intervals showed good healing of the bladder wall and no recurrence of the tumor. Discussion Single intravesical instillation with mitomycin C (MMC), epirubicin, or pirarubicin, have shown a beneficial effect in reducing recurrence rate compared to TURBT alone. However, randomized comparisons of individual drugs have not been conducted.3 The high recurrence rate of NMIBC may be attributed to the seeding of floating tumor cells. Immediate chemotherapy instillation has been shown to act by destroying these circulating tumor cells after TURBT. Hence the EAU guidelines recommend initiating the instillation within the first few hours after TURBT.1 Most of the complications of intravesical chemotherapy instillation are minor and can be managed medically. Dysuria and urinary frequency and urgency were the most common complications.4 On the other hand, reporting major complications such as bladder perforation, perirectal abscesses, eosinophilic cystitis, and ureteral stenosis is limited to case reports.5-14 In this case, we described our experience with bladder perforation after immediate intravesical instillation of doxorubicin. One can argue that the perforation has occurred intraoperatively and gone unnoticed. However, in view of the absence of endoscopic evidence of perforation, late presentation of symptoms, and the absence of symptoms during and after the instillation we assume that the Single Dose Intravesical Doxorubicin Instillation caused the perforation rather than complicating an existing one. To explain the mechanism of perforation, Lim et al7 and Cliff et al8 hypothesized that perforation is caused when attenuated muscularis propria gets necrosed after MMC instillation. The presenting complaints of our patient were perineal and pelvic pain as well as urinary irritative symptoms. This is similar to Elmamoun et al9 cohort in which all the 6 patients presented with perineal and pelvic pain. Other symptoms such as fever, urine retention, and recurrent UTI were variably reported by others. Table 1 summarizes the clinical presentation and management of bladder perforation in the previously published case reports. Table1. A summary of the presentation and management of bladder perforation after immediate chemotheraputic agent intravesical instillation. Ref. number The instilled drug Number of patients Initial presentation Management outline Lim et al7 MMC 1 Pelvic and suprapubic pain Exploratry laparotomy and bladder repair after failed conservative management Elmamoun et al9 MMC 6 3 patients presented with pelvic pain Conservative management and urethral catheterization for 2, 26, and 52 weeks Pelvic pain Recurrent UTIs Left ureteric obstruction Partial cystectomy + left to right trans-ureteroureterostomy Pelvic pain, Persistent urinary leak Left ureteric obstruction Augmentation cystoplasty + leftUreteric reimplantationThe patient ended with persistent leak, so a long-term catheter was used Pelvic painSevere LUTS Bilateral ureteric obstruction Urinary diversion (ileal conduit) Penna et al10 MMC 1 Lower abdominal pain and inability to void Analgesia, antibiotics and urethral catheter for 4 weeks Tyritzis et al11 EPI 2 Fever and retropubic pain Conservative: Antibiotics and urethral catheter Fever, lower abdominal pain and cellulitis Exploratry laparotomy and bladder repair after failed conservative management Nieuwenhuijzen et al12 MMC Pelvic pain and fever Exploratry laparotomy and bladder repair after failed conservative management for 1 month Racioppi et al13 MMC Pelvic pain, fever and pancytopenia Exploratry laparotomy and bladder repair after failed conservative management for 3 weeks Hatem and Leifeld14 MMC Pelvic pain Fever and positive blood culture for ecoli as well as purulent discharge from the drain site after the first expolratory laparatomy Conservative management for 10 daysExploratory laparatomy that revealed edematous anterior bladder wall without defectRenewed laparotomy, drainage of the abscess, debridement of the necrotic tissue, mono-J ureteral stents and nephrostomyRadical cystectomy with a urinary diversion through ureterocutaneostomy Shenaq et al15 EPI 3 Strong urge and pain in the lower left abdomen Conservative management with antibiotics and urethral catheterization Abdominal pain and intestinal obstruction Conservative management for 12 daysExploratory laparotomy that revealed infiltrative mass on the top of the bladder causing IO, so colostomy was done.The patient died due to multi organ failure Progressive pain in the lower abdomen Conservative management with antibiotics and urethral catheterization Abbreviations: Ep, epirubicin; MMC, mitomycin C. Concerning the management, almost all the reported cases were initially managed conservatively by urethral catheterization, anticholinergics, and antibiotics. However, this was insufficient to heal the bladder in some cases which needed open repair. Moreover, some ended with cystectomy. In our case, the patient has an extraperitoneal extravasation that failed the conservative management and needed open surgical repair. A striking feature of the healing process that was seen in many cases including ours is the poor and slow healing. In our case, the patient needed 9 weeks for complete healing after proper open 3 layers bladder wall repair.7-9,13,14 While Lim et al attributed the slow healing in their case to the patient’s pre-existing peripheral vascular disease and suboptimal tissue oxygenation, no risk factors were identified in the other cases including our patient who was free of any comorbidities. We suppose that the poor healing is linked to the chemotherapeutic agent inflammatory effect rather than to patient factor. This assumption is backed by the reported characteristic of mitomycin induced skin ulcer in cases of extravasation after intravenous administration. These ulcers are well known for their chronicity and the low tendency of spontaneous healing. Moreover, debridement and soft-tissue reconstruction following extravasation of MMC are recommended for patients with persistent ulceration and pain.15 Another common finding in the cases that mandate open repair was the presence of perivesical soft tissue necrosis and extensive inflammation. Doherty et al observed that more extensive bladder wall and fat necrosis of extravesical tissue in cystectomy specimens occurred when either epirubicin or mitomycin instillation was administered than in that seen following TURBT alone.16 This cytotoxic effect was also reported by Tyritzis et al11 after epirubicin instillation, and by Cliff et al,8 Nieuwenhuijzen et al,12 and Hatem and Leifeld14 after MMC instillation. This cytotoxic effect was recognized herein for the doxorubicin instillation, which is the first time to be reported as a complication of doxorubicin. It is worth to mention that the perforation had no adverse oncological effect on our patient as no tumor recurrence was detected during follow up. Conclusion Immediate intravesical instillations of chemotherapy are not devoid from risk and serious complications as bladder perforation can occur. Whether it’s value in decreasing the recurrence justify the risk of bladder perforation is not our argument in this case report. Nevertheless, this severe complication should be kept in mind, and high index of suspicion will aid in timely management. If perforation is suspected, urethral catheterization and contrasted imaging are mandatory. In the case of failed conservative management surgical repair is warranted even in extraperitoneal leak. Funding:The author(s) received no financial support for the research, authorship, and/or publication of this article. Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Author Contributions: Concept: AA, SAD. Design: MA, SA. Supervision: AA, SAD, MAA. Resources: MA, HM, NB. Materials: AA, MA, NB. Writing manuscript: AA, MA, HM. Critical review: AA, SAD, MAA. Informed Consent: The patient has given his written informed consent to publish his case including publication of images. The study follows the guidelines for human studies and is conducted in accordance with the World Medical Association Declaration of Helsinki. ORCID iD: Hammam Mansi https://orcid.org/0000-0002-4571-4336	DOXORUBICIN HYDROCHLORIDE	DrugsGivenReaction	CC BY-NC	33473244	18,802,244	2021
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Pelvic pain'.	Delayed Bladder Perforation Related to Immediate Single Dose Intravesical Doxorubicin Instillation After TURBT: A Case Report and Literature Review. Intravesical chemotherapy instillation immediately after tumor resection is a well-known practice in the management of non-muscle invasive bladder cancer. Despite being largely well tolerated in most cases, it is not devoid of severe and life-threatening complications. We present an unusual case of bladder perforation that happened 2 weeks after bladder tumor resection. The patient had received single dose intra-vesical instillation of doxorubicin after TUR-BT. Conservative managements failed to achieve bladder healing; as a result, open surgical repair was performed. To the best of our knowledge, this is the first reported case of bladder perforation after intra-vesical doxorubicin instillation. The occurrence of such a rare serious complication in a mostly safe intervention must be taken into consideration. A high index of suspicion, timely management, and proceeding to more invasive surgical treatments when necessary are cornerstones in the management and preserving the bladder. Introduction Bladder cancer (BC) is the seventh most commonly diagnosed cancer in men worldwide. About 75% of the cases are Non-muscle invasive (NMIBC).1 Transurethral resection of the bladder tumor (TURBT) remains the gold standard of therapy for diagnosing and treating NMIBC. To decrease the recurrence rate of low-risk tumors, a single, immediate, post-operative intravesical instillation of chemotherapy is recommended.1 The most commonly used chemotherapeutic agent is Mitomycin C (MMC), which is an alkylating agent that inhibits DNA synthesis and causes single-strand breakage of DNA and chromosomal breaks.2 Immediate single instillation (SI) is largely well-tolerated. However, severe complications and even death have been reported in the literature. Here we present a case wherein the patient had an extraperitoneal bladder perforation that occurred 2 weeks after TUT-BT and immediate postoperative instillation of doxorubicin. Case Presentation A 63-Year-old male, a heavy smoker, with no other co-morbidities, was referred to urology clinic for evaluation and management of a lesion on the left bladder wall that was incidentally detected on a CT scan. He did not have any urinary symptoms on initial presentation. Cystourethroscopy revealed 2 papillary growths on the left lateral bladder wall, which were resected completely and smoothly by a resectoscope using a monopolar energy. Complete resection of the tumor was achieved without any intra-operative complication or perforation. Hemostasis was secured, and a urethral catheter was inserted. In accordance of the EAU guidelines, the patient received an intravesical instillation of 50 ml of doxorubicin diluted in 100 cc of saline 6 hours after the TURBT. During the instillation, his vital signs were stable and he had neither hematuria nor abdominal pain. One hour after the instillation, the catheter was unclamped and was attached to continuous bladder irrigation. Two days after the surgery and in the absence of hematuria, the indwelling catheter was removed, and the patient was discharged home the next day. When seen in the clinic 1 week later the patient was fine with no alarming symptoms. The pathology report showed a low-grade Ta Papillary urothelial carcinoma. Based on the pathological diagnosis and according to the EAU guidelines, adjuvant chemotherapy instillations were planned. Two weeks later, the patient presented with, dysuria, frequency, urgency, as well as pelvic and perineal pain. Physical examination was unremarkable. Urine analysis showed numerous WBC’s, RBC’s, and bacteria. An empirical antibiotics course was commenced. In view of the worsening symptoms and the past history of anal fistula, a contrast enhanced pelvic MRI was performed. It showed a diffusely thickened urinary bladder wall, with evidence of a focal wall defect at its left aspect (the site of the resected tumor). The active urine leak into the perivesical space confirmed the diagnosis of retroperitoneal bladder perforation (Figure 1). Figure. 1. Pelvic MRI (axial view) showing extraperitoneal bladder perforation at the left lateral wall (arrow points to extravasated contrast). The patient was managed conservatively with antibiotics and an indwelling catheter for 3 weeks. A follow-up retrograde CT cystogram showed no improvement and a gross contrast leak from the same site was identified. The catheter was kept for 6 more weeks and the CT cystogram was repeated showing the same results with no evidence of improvement. The patient refused to keep the catheter for a longer period so the option of open surgical repair was discussed with him. Extraperitoneal laparotomy was performed, a perivesical small abscess was drained and cultured, necrotic tissues were debrided, and the bladder wall defect was rimed and closed in 3 layers. Tissue biopsies from the pelvic wall and bladder wall were harvested. A urethral catheter and pelvic drain were kept in place. The postoperative course was uneventful, the drain was removed on the third-day postoperatively, and the patient was discharged home with the urethral catheter in. Histopathological evaluation of the pelvic wall and the bladder biopsies verified acute and chronic inflammation as well as granulation tissue formation. However, both were negative for malignancy. The urinary bladder biopsy also showed extensive foci of dystrophic calcification while the pelvic wall biopsy revealed fat necrosis (Figures 2 and 3). Figure 2. The histologic examination revealed fragments of soft tissue with fat necrosis in the lower left area, mixed acute and chronic inflammatory cell infiltrate and hemorrhage in the upper right area (Hematoxylin & Eosin, 200X). Figure 3. The histologic examination revealed multiple areas of calcifications embedded deep in the tissue with associated crystal-like material suggestive of calcium-oxalate or talc powder crystals (Hematoxylin & Eosin, 200X). A follow up CT cystogram was done 3 weeks after the bladder repair. Although it showed a considerable regression in the size of the bladder defect, interestingly it also showed persistent extra-peritoneal contrast leak from the bladder along it’s left lateral aspect (Figure 4). Figure 4. Retrograde Ct cystogram showing persistent contrast leak from the left lateral aspect after surgical repair. Due to this slow healing, the catheter was kept in. Six weeks later, a follow up CT cystogram showed healing with no extraperitoneal contrast leak (Figure 5). And hence the catheter was removed. Figure 5. Retrograde Ct cystogram showing a healed bladder wall with no contrast leak. Two follow up cystoscopies and bladder biopsy (from an erythematous area on the left lateral wall) with 3 months intervals showed good healing of the bladder wall and no recurrence of the tumor. Discussion Single intravesical instillation with mitomycin C (MMC), epirubicin, or pirarubicin, have shown a beneficial effect in reducing recurrence rate compared to TURBT alone. However, randomized comparisons of individual drugs have not been conducted.3 The high recurrence rate of NMIBC may be attributed to the seeding of floating tumor cells. Immediate chemotherapy instillation has been shown to act by destroying these circulating tumor cells after TURBT. Hence the EAU guidelines recommend initiating the instillation within the first few hours after TURBT.1 Most of the complications of intravesical chemotherapy instillation are minor and can be managed medically. Dysuria and urinary frequency and urgency were the most common complications.4 On the other hand, reporting major complications such as bladder perforation, perirectal abscesses, eosinophilic cystitis, and ureteral stenosis is limited to case reports.5-14 In this case, we described our experience with bladder perforation after immediate intravesical instillation of doxorubicin. One can argue that the perforation has occurred intraoperatively and gone unnoticed. However, in view of the absence of endoscopic evidence of perforation, late presentation of symptoms, and the absence of symptoms during and after the instillation we assume that the Single Dose Intravesical Doxorubicin Instillation caused the perforation rather than complicating an existing one. To explain the mechanism of perforation, Lim et al7 and Cliff et al8 hypothesized that perforation is caused when attenuated muscularis propria gets necrosed after MMC instillation. The presenting complaints of our patient were perineal and pelvic pain as well as urinary irritative symptoms. This is similar to Elmamoun et al9 cohort in which all the 6 patients presented with perineal and pelvic pain. Other symptoms such as fever, urine retention, and recurrent UTI were variably reported by others. Table 1 summarizes the clinical presentation and management of bladder perforation in the previously published case reports. Table1. A summary of the presentation and management of bladder perforation after immediate chemotheraputic agent intravesical instillation. Ref. number The instilled drug Number of patients Initial presentation Management outline Lim et al7 MMC 1 Pelvic and suprapubic pain Exploratry laparotomy and bladder repair after failed conservative management Elmamoun et al9 MMC 6 3 patients presented with pelvic pain Conservative management and urethral catheterization for 2, 26, and 52 weeks Pelvic pain Recurrent UTIs Left ureteric obstruction Partial cystectomy + left to right trans-ureteroureterostomy Pelvic pain, Persistent urinary leak Left ureteric obstruction Augmentation cystoplasty + leftUreteric reimplantationThe patient ended with persistent leak, so a long-term catheter was used Pelvic painSevere LUTS Bilateral ureteric obstruction Urinary diversion (ileal conduit) Penna et al10 MMC 1 Lower abdominal pain and inability to void Analgesia, antibiotics and urethral catheter for 4 weeks Tyritzis et al11 EPI 2 Fever and retropubic pain Conservative: Antibiotics and urethral catheter Fever, lower abdominal pain and cellulitis Exploratry laparotomy and bladder repair after failed conservative management Nieuwenhuijzen et al12 MMC Pelvic pain and fever Exploratry laparotomy and bladder repair after failed conservative management for 1 month Racioppi et al13 MMC Pelvic pain, fever and pancytopenia Exploratry laparotomy and bladder repair after failed conservative management for 3 weeks Hatem and Leifeld14 MMC Pelvic pain Fever and positive blood culture for ecoli as well as purulent discharge from the drain site after the first expolratory laparatomy Conservative management for 10 daysExploratory laparatomy that revealed edematous anterior bladder wall without defectRenewed laparotomy, drainage of the abscess, debridement of the necrotic tissue, mono-J ureteral stents and nephrostomyRadical cystectomy with a urinary diversion through ureterocutaneostomy Shenaq et al15 EPI 3 Strong urge and pain in the lower left abdomen Conservative management with antibiotics and urethral catheterization Abdominal pain and intestinal obstruction Conservative management for 12 daysExploratory laparotomy that revealed infiltrative mass on the top of the bladder causing IO, so colostomy was done.The patient died due to multi organ failure Progressive pain in the lower abdomen Conservative management with antibiotics and urethral catheterization Abbreviations: Ep, epirubicin; MMC, mitomycin C. Concerning the management, almost all the reported cases were initially managed conservatively by urethral catheterization, anticholinergics, and antibiotics. However, this was insufficient to heal the bladder in some cases which needed open repair. Moreover, some ended with cystectomy. In our case, the patient has an extraperitoneal extravasation that failed the conservative management and needed open surgical repair. A striking feature of the healing process that was seen in many cases including ours is the poor and slow healing. In our case, the patient needed 9 weeks for complete healing after proper open 3 layers bladder wall repair.7-9,13,14 While Lim et al attributed the slow healing in their case to the patient’s pre-existing peripheral vascular disease and suboptimal tissue oxygenation, no risk factors were identified in the other cases including our patient who was free of any comorbidities. We suppose that the poor healing is linked to the chemotherapeutic agent inflammatory effect rather than to patient factor. This assumption is backed by the reported characteristic of mitomycin induced skin ulcer in cases of extravasation after intravenous administration. These ulcers are well known for their chronicity and the low tendency of spontaneous healing. Moreover, debridement and soft-tissue reconstruction following extravasation of MMC are recommended for patients with persistent ulceration and pain.15 Another common finding in the cases that mandate open repair was the presence of perivesical soft tissue necrosis and extensive inflammation. Doherty et al observed that more extensive bladder wall and fat necrosis of extravesical tissue in cystectomy specimens occurred when either epirubicin or mitomycin instillation was administered than in that seen following TURBT alone.16 This cytotoxic effect was also reported by Tyritzis et al11 after epirubicin instillation, and by Cliff et al,8 Nieuwenhuijzen et al,12 and Hatem and Leifeld14 after MMC instillation. This cytotoxic effect was recognized herein for the doxorubicin instillation, which is the first time to be reported as a complication of doxorubicin. It is worth to mention that the perforation had no adverse oncological effect on our patient as no tumor recurrence was detected during follow up. Conclusion Immediate intravesical instillations of chemotherapy are not devoid from risk and serious complications as bladder perforation can occur. Whether it’s value in decreasing the recurrence justify the risk of bladder perforation is not our argument in this case report. Nevertheless, this severe complication should be kept in mind, and high index of suspicion will aid in timely management. If perforation is suspected, urethral catheterization and contrasted imaging are mandatory. In the case of failed conservative management surgical repair is warranted even in extraperitoneal leak. Funding:The author(s) received no financial support for the research, authorship, and/or publication of this article. Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Author Contributions: Concept: AA, SAD. Design: MA, SA. Supervision: AA, SAD, MAA. Resources: MA, HM, NB. Materials: AA, MA, NB. Writing manuscript: AA, MA, HM. Critical review: AA, SAD, MAA. Informed Consent: The patient has given his written informed consent to publish his case including publication of images. The study follows the guidelines for human studies and is conducted in accordance with the World Medical Association Declaration of Helsinki. ORCID iD: Hammam Mansi https://orcid.org/0000-0002-4571-4336	DOXORUBICIN	DrugsGivenReaction	CC BY-NC	33473244	18,870,263	2021
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Perineal pain'.	Delayed Bladder Perforation Related to Immediate Single Dose Intravesical Doxorubicin Instillation After TURBT: A Case Report and Literature Review. Intravesical chemotherapy instillation immediately after tumor resection is a well-known practice in the management of non-muscle invasive bladder cancer. Despite being largely well tolerated in most cases, it is not devoid of severe and life-threatening complications. We present an unusual case of bladder perforation that happened 2 weeks after bladder tumor resection. The patient had received single dose intra-vesical instillation of doxorubicin after TUR-BT. Conservative managements failed to achieve bladder healing; as a result, open surgical repair was performed. To the best of our knowledge, this is the first reported case of bladder perforation after intra-vesical doxorubicin instillation. The occurrence of such a rare serious complication in a mostly safe intervention must be taken into consideration. A high index of suspicion, timely management, and proceeding to more invasive surgical treatments when necessary are cornerstones in the management and preserving the bladder. Introduction Bladder cancer (BC) is the seventh most commonly diagnosed cancer in men worldwide. About 75% of the cases are Non-muscle invasive (NMIBC).1 Transurethral resection of the bladder tumor (TURBT) remains the gold standard of therapy for diagnosing and treating NMIBC. To decrease the recurrence rate of low-risk tumors, a single, immediate, post-operative intravesical instillation of chemotherapy is recommended.1 The most commonly used chemotherapeutic agent is Mitomycin C (MMC), which is an alkylating agent that inhibits DNA synthesis and causes single-strand breakage of DNA and chromosomal breaks.2 Immediate single instillation (SI) is largely well-tolerated. However, severe complications and even death have been reported in the literature. Here we present a case wherein the patient had an extraperitoneal bladder perforation that occurred 2 weeks after TUT-BT and immediate postoperative instillation of doxorubicin. Case Presentation A 63-Year-old male, a heavy smoker, with no other co-morbidities, was referred to urology clinic for evaluation and management of a lesion on the left bladder wall that was incidentally detected on a CT scan. He did not have any urinary symptoms on initial presentation. Cystourethroscopy revealed 2 papillary growths on the left lateral bladder wall, which were resected completely and smoothly by a resectoscope using a monopolar energy. Complete resection of the tumor was achieved without any intra-operative complication or perforation. Hemostasis was secured, and a urethral catheter was inserted. In accordance of the EAU guidelines, the patient received an intravesical instillation of 50 ml of doxorubicin diluted in 100 cc of saline 6 hours after the TURBT. During the instillation, his vital signs were stable and he had neither hematuria nor abdominal pain. One hour after the instillation, the catheter was unclamped and was attached to continuous bladder irrigation. Two days after the surgery and in the absence of hematuria, the indwelling catheter was removed, and the patient was discharged home the next day. When seen in the clinic 1 week later the patient was fine with no alarming symptoms. The pathology report showed a low-grade Ta Papillary urothelial carcinoma. Based on the pathological diagnosis and according to the EAU guidelines, adjuvant chemotherapy instillations were planned. Two weeks later, the patient presented with, dysuria, frequency, urgency, as well as pelvic and perineal pain. Physical examination was unremarkable. Urine analysis showed numerous WBC’s, RBC’s, and bacteria. An empirical antibiotics course was commenced. In view of the worsening symptoms and the past history of anal fistula, a contrast enhanced pelvic MRI was performed. It showed a diffusely thickened urinary bladder wall, with evidence of a focal wall defect at its left aspect (the site of the resected tumor). The active urine leak into the perivesical space confirmed the diagnosis of retroperitoneal bladder perforation (Figure 1). Figure. 1. Pelvic MRI (axial view) showing extraperitoneal bladder perforation at the left lateral wall (arrow points to extravasated contrast). The patient was managed conservatively with antibiotics and an indwelling catheter for 3 weeks. A follow-up retrograde CT cystogram showed no improvement and a gross contrast leak from the same site was identified. The catheter was kept for 6 more weeks and the CT cystogram was repeated showing the same results with no evidence of improvement. The patient refused to keep the catheter for a longer period so the option of open surgical repair was discussed with him. Extraperitoneal laparotomy was performed, a perivesical small abscess was drained and cultured, necrotic tissues were debrided, and the bladder wall defect was rimed and closed in 3 layers. Tissue biopsies from the pelvic wall and bladder wall were harvested. A urethral catheter and pelvic drain were kept in place. The postoperative course was uneventful, the drain was removed on the third-day postoperatively, and the patient was discharged home with the urethral catheter in. Histopathological evaluation of the pelvic wall and the bladder biopsies verified acute and chronic inflammation as well as granulation tissue formation. However, both were negative for malignancy. The urinary bladder biopsy also showed extensive foci of dystrophic calcification while the pelvic wall biopsy revealed fat necrosis (Figures 2 and 3). Figure 2. The histologic examination revealed fragments of soft tissue with fat necrosis in the lower left area, mixed acute and chronic inflammatory cell infiltrate and hemorrhage in the upper right area (Hematoxylin & Eosin, 200X). Figure 3. The histologic examination revealed multiple areas of calcifications embedded deep in the tissue with associated crystal-like material suggestive of calcium-oxalate or talc powder crystals (Hematoxylin & Eosin, 200X). A follow up CT cystogram was done 3 weeks after the bladder repair. Although it showed a considerable regression in the size of the bladder defect, interestingly it also showed persistent extra-peritoneal contrast leak from the bladder along it’s left lateral aspect (Figure 4). Figure 4. Retrograde Ct cystogram showing persistent contrast leak from the left lateral aspect after surgical repair. Due to this slow healing, the catheter was kept in. Six weeks later, a follow up CT cystogram showed healing with no extraperitoneal contrast leak (Figure 5). And hence the catheter was removed. Figure 5. Retrograde Ct cystogram showing a healed bladder wall with no contrast leak. Two follow up cystoscopies and bladder biopsy (from an erythematous area on the left lateral wall) with 3 months intervals showed good healing of the bladder wall and no recurrence of the tumor. Discussion Single intravesical instillation with mitomycin C (MMC), epirubicin, or pirarubicin, have shown a beneficial effect in reducing recurrence rate compared to TURBT alone. However, randomized comparisons of individual drugs have not been conducted.3 The high recurrence rate of NMIBC may be attributed to the seeding of floating tumor cells. Immediate chemotherapy instillation has been shown to act by destroying these circulating tumor cells after TURBT. Hence the EAU guidelines recommend initiating the instillation within the first few hours after TURBT.1 Most of the complications of intravesical chemotherapy instillation are minor and can be managed medically. Dysuria and urinary frequency and urgency were the most common complications.4 On the other hand, reporting major complications such as bladder perforation, perirectal abscesses, eosinophilic cystitis, and ureteral stenosis is limited to case reports.5-14 In this case, we described our experience with bladder perforation after immediate intravesical instillation of doxorubicin. One can argue that the perforation has occurred intraoperatively and gone unnoticed. However, in view of the absence of endoscopic evidence of perforation, late presentation of symptoms, and the absence of symptoms during and after the instillation we assume that the Single Dose Intravesical Doxorubicin Instillation caused the perforation rather than complicating an existing one. To explain the mechanism of perforation, Lim et al7 and Cliff et al8 hypothesized that perforation is caused when attenuated muscularis propria gets necrosed after MMC instillation. The presenting complaints of our patient were perineal and pelvic pain as well as urinary irritative symptoms. This is similar to Elmamoun et al9 cohort in which all the 6 patients presented with perineal and pelvic pain. Other symptoms such as fever, urine retention, and recurrent UTI were variably reported by others. Table 1 summarizes the clinical presentation and management of bladder perforation in the previously published case reports. Table1. A summary of the presentation and management of bladder perforation after immediate chemotheraputic agent intravesical instillation. Ref. number The instilled drug Number of patients Initial presentation Management outline Lim et al7 MMC 1 Pelvic and suprapubic pain Exploratry laparotomy and bladder repair after failed conservative management Elmamoun et al9 MMC 6 3 patients presented with pelvic pain Conservative management and urethral catheterization for 2, 26, and 52 weeks Pelvic pain Recurrent UTIs Left ureteric obstruction Partial cystectomy + left to right trans-ureteroureterostomy Pelvic pain, Persistent urinary leak Left ureteric obstruction Augmentation cystoplasty + leftUreteric reimplantationThe patient ended with persistent leak, so a long-term catheter was used Pelvic painSevere LUTS Bilateral ureteric obstruction Urinary diversion (ileal conduit) Penna et al10 MMC 1 Lower abdominal pain and inability to void Analgesia, antibiotics and urethral catheter for 4 weeks Tyritzis et al11 EPI 2 Fever and retropubic pain Conservative: Antibiotics and urethral catheter Fever, lower abdominal pain and cellulitis Exploratry laparotomy and bladder repair after failed conservative management Nieuwenhuijzen et al12 MMC Pelvic pain and fever Exploratry laparotomy and bladder repair after failed conservative management for 1 month Racioppi et al13 MMC Pelvic pain, fever and pancytopenia Exploratry laparotomy and bladder repair after failed conservative management for 3 weeks Hatem and Leifeld14 MMC Pelvic pain Fever and positive blood culture for ecoli as well as purulent discharge from the drain site after the first expolratory laparatomy Conservative management for 10 daysExploratory laparatomy that revealed edematous anterior bladder wall without defectRenewed laparotomy, drainage of the abscess, debridement of the necrotic tissue, mono-J ureteral stents and nephrostomyRadical cystectomy with a urinary diversion through ureterocutaneostomy Shenaq et al15 EPI 3 Strong urge and pain in the lower left abdomen Conservative management with antibiotics and urethral catheterization Abdominal pain and intestinal obstruction Conservative management for 12 daysExploratory laparotomy that revealed infiltrative mass on the top of the bladder causing IO, so colostomy was done.The patient died due to multi organ failure Progressive pain in the lower abdomen Conservative management with antibiotics and urethral catheterization Abbreviations: Ep, epirubicin; MMC, mitomycin C. Concerning the management, almost all the reported cases were initially managed conservatively by urethral catheterization, anticholinergics, and antibiotics. However, this was insufficient to heal the bladder in some cases which needed open repair. Moreover, some ended with cystectomy. In our case, the patient has an extraperitoneal extravasation that failed the conservative management and needed open surgical repair. A striking feature of the healing process that was seen in many cases including ours is the poor and slow healing. In our case, the patient needed 9 weeks for complete healing after proper open 3 layers bladder wall repair.7-9,13,14 While Lim et al attributed the slow healing in their case to the patient’s pre-existing peripheral vascular disease and suboptimal tissue oxygenation, no risk factors were identified in the other cases including our patient who was free of any comorbidities. We suppose that the poor healing is linked to the chemotherapeutic agent inflammatory effect rather than to patient factor. This assumption is backed by the reported characteristic of mitomycin induced skin ulcer in cases of extravasation after intravenous administration. These ulcers are well known for their chronicity and the low tendency of spontaneous healing. Moreover, debridement and soft-tissue reconstruction following extravasation of MMC are recommended for patients with persistent ulceration and pain.15 Another common finding in the cases that mandate open repair was the presence of perivesical soft tissue necrosis and extensive inflammation. Doherty et al observed that more extensive bladder wall and fat necrosis of extravesical tissue in cystectomy specimens occurred when either epirubicin or mitomycin instillation was administered than in that seen following TURBT alone.16 This cytotoxic effect was also reported by Tyritzis et al11 after epirubicin instillation, and by Cliff et al,8 Nieuwenhuijzen et al,12 and Hatem and Leifeld14 after MMC instillation. This cytotoxic effect was recognized herein for the doxorubicin instillation, which is the first time to be reported as a complication of doxorubicin. It is worth to mention that the perforation had no adverse oncological effect on our patient as no tumor recurrence was detected during follow up. Conclusion Immediate intravesical instillations of chemotherapy are not devoid from risk and serious complications as bladder perforation can occur. Whether it’s value in decreasing the recurrence justify the risk of bladder perforation is not our argument in this case report. Nevertheless, this severe complication should be kept in mind, and high index of suspicion will aid in timely management. If perforation is suspected, urethral catheterization and contrasted imaging are mandatory. In the case of failed conservative management surgical repair is warranted even in extraperitoneal leak. Funding:The author(s) received no financial support for the research, authorship, and/or publication of this article. Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Author Contributions: Concept: AA, SAD. Design: MA, SA. Supervision: AA, SAD, MAA. Resources: MA, HM, NB. Materials: AA, MA, NB. Writing manuscript: AA, MA, HM. Critical review: AA, SAD, MAA. Informed Consent: The patient has given his written informed consent to publish his case including publication of images. The study follows the guidelines for human studies and is conducted in accordance with the World Medical Association Declaration of Helsinki. ORCID iD: Hammam Mansi https://orcid.org/0000-0002-4571-4336	DOXORUBICIN	DrugsGivenReaction	CC BY-NC	33473244	18,870,263	2021
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Pollakiuria'.	Delayed Bladder Perforation Related to Immediate Single Dose Intravesical Doxorubicin Instillation After TURBT: A Case Report and Literature Review. Intravesical chemotherapy instillation immediately after tumor resection is a well-known practice in the management of non-muscle invasive bladder cancer. Despite being largely well tolerated in most cases, it is not devoid of severe and life-threatening complications. We present an unusual case of bladder perforation that happened 2 weeks after bladder tumor resection. The patient had received single dose intra-vesical instillation of doxorubicin after TUR-BT. Conservative managements failed to achieve bladder healing; as a result, open surgical repair was performed. To the best of our knowledge, this is the first reported case of bladder perforation after intra-vesical doxorubicin instillation. The occurrence of such a rare serious complication in a mostly safe intervention must be taken into consideration. A high index of suspicion, timely management, and proceeding to more invasive surgical treatments when necessary are cornerstones in the management and preserving the bladder. Introduction Bladder cancer (BC) is the seventh most commonly diagnosed cancer in men worldwide. About 75% of the cases are Non-muscle invasive (NMIBC).1 Transurethral resection of the bladder tumor (TURBT) remains the gold standard of therapy for diagnosing and treating NMIBC. To decrease the recurrence rate of low-risk tumors, a single, immediate, post-operative intravesical instillation of chemotherapy is recommended.1 The most commonly used chemotherapeutic agent is Mitomycin C (MMC), which is an alkylating agent that inhibits DNA synthesis and causes single-strand breakage of DNA and chromosomal breaks.2 Immediate single instillation (SI) is largely well-tolerated. However, severe complications and even death have been reported in the literature. Here we present a case wherein the patient had an extraperitoneal bladder perforation that occurred 2 weeks after TUT-BT and immediate postoperative instillation of doxorubicin. Case Presentation A 63-Year-old male, a heavy smoker, with no other co-morbidities, was referred to urology clinic for evaluation and management of a lesion on the left bladder wall that was incidentally detected on a CT scan. He did not have any urinary symptoms on initial presentation. Cystourethroscopy revealed 2 papillary growths on the left lateral bladder wall, which were resected completely and smoothly by a resectoscope using a monopolar energy. Complete resection of the tumor was achieved without any intra-operative complication or perforation. Hemostasis was secured, and a urethral catheter was inserted. In accordance of the EAU guidelines, the patient received an intravesical instillation of 50 ml of doxorubicin diluted in 100 cc of saline 6 hours after the TURBT. During the instillation, his vital signs were stable and he had neither hematuria nor abdominal pain. One hour after the instillation, the catheter was unclamped and was attached to continuous bladder irrigation. Two days after the surgery and in the absence of hematuria, the indwelling catheter was removed, and the patient was discharged home the next day. When seen in the clinic 1 week later the patient was fine with no alarming symptoms. The pathology report showed a low-grade Ta Papillary urothelial carcinoma. Based on the pathological diagnosis and according to the EAU guidelines, adjuvant chemotherapy instillations were planned. Two weeks later, the patient presented with, dysuria, frequency, urgency, as well as pelvic and perineal pain. Physical examination was unremarkable. Urine analysis showed numerous WBC’s, RBC’s, and bacteria. An empirical antibiotics course was commenced. In view of the worsening symptoms and the past history of anal fistula, a contrast enhanced pelvic MRI was performed. It showed a diffusely thickened urinary bladder wall, with evidence of a focal wall defect at its left aspect (the site of the resected tumor). The active urine leak into the perivesical space confirmed the diagnosis of retroperitoneal bladder perforation (Figure 1). Figure. 1. Pelvic MRI (axial view) showing extraperitoneal bladder perforation at the left lateral wall (arrow points to extravasated contrast). The patient was managed conservatively with antibiotics and an indwelling catheter for 3 weeks. A follow-up retrograde CT cystogram showed no improvement and a gross contrast leak from the same site was identified. The catheter was kept for 6 more weeks and the CT cystogram was repeated showing the same results with no evidence of improvement. The patient refused to keep the catheter for a longer period so the option of open surgical repair was discussed with him. Extraperitoneal laparotomy was performed, a perivesical small abscess was drained and cultured, necrotic tissues were debrided, and the bladder wall defect was rimed and closed in 3 layers. Tissue biopsies from the pelvic wall and bladder wall were harvested. A urethral catheter and pelvic drain were kept in place. The postoperative course was uneventful, the drain was removed on the third-day postoperatively, and the patient was discharged home with the urethral catheter in. Histopathological evaluation of the pelvic wall and the bladder biopsies verified acute and chronic inflammation as well as granulation tissue formation. However, both were negative for malignancy. The urinary bladder biopsy also showed extensive foci of dystrophic calcification while the pelvic wall biopsy revealed fat necrosis (Figures 2 and 3). Figure 2. The histologic examination revealed fragments of soft tissue with fat necrosis in the lower left area, mixed acute and chronic inflammatory cell infiltrate and hemorrhage in the upper right area (Hematoxylin & Eosin, 200X). Figure 3. The histologic examination revealed multiple areas of calcifications embedded deep in the tissue with associated crystal-like material suggestive of calcium-oxalate or talc powder crystals (Hematoxylin & Eosin, 200X). A follow up CT cystogram was done 3 weeks after the bladder repair. Although it showed a considerable regression in the size of the bladder defect, interestingly it also showed persistent extra-peritoneal contrast leak from the bladder along it’s left lateral aspect (Figure 4). Figure 4. Retrograde Ct cystogram showing persistent contrast leak from the left lateral aspect after surgical repair. Due to this slow healing, the catheter was kept in. Six weeks later, a follow up CT cystogram showed healing with no extraperitoneal contrast leak (Figure 5). And hence the catheter was removed. Figure 5. Retrograde Ct cystogram showing a healed bladder wall with no contrast leak. Two follow up cystoscopies and bladder biopsy (from an erythematous area on the left lateral wall) with 3 months intervals showed good healing of the bladder wall and no recurrence of the tumor. Discussion Single intravesical instillation with mitomycin C (MMC), epirubicin, or pirarubicin, have shown a beneficial effect in reducing recurrence rate compared to TURBT alone. However, randomized comparisons of individual drugs have not been conducted.3 The high recurrence rate of NMIBC may be attributed to the seeding of floating tumor cells. Immediate chemotherapy instillation has been shown to act by destroying these circulating tumor cells after TURBT. Hence the EAU guidelines recommend initiating the instillation within the first few hours after TURBT.1 Most of the complications of intravesical chemotherapy instillation are minor and can be managed medically. Dysuria and urinary frequency and urgency were the most common complications.4 On the other hand, reporting major complications such as bladder perforation, perirectal abscesses, eosinophilic cystitis, and ureteral stenosis is limited to case reports.5-14 In this case, we described our experience with bladder perforation after immediate intravesical instillation of doxorubicin. One can argue that the perforation has occurred intraoperatively and gone unnoticed. However, in view of the absence of endoscopic evidence of perforation, late presentation of symptoms, and the absence of symptoms during and after the instillation we assume that the Single Dose Intravesical Doxorubicin Instillation caused the perforation rather than complicating an existing one. To explain the mechanism of perforation, Lim et al7 and Cliff et al8 hypothesized that perforation is caused when attenuated muscularis propria gets necrosed after MMC instillation. The presenting complaints of our patient were perineal and pelvic pain as well as urinary irritative symptoms. This is similar to Elmamoun et al9 cohort in which all the 6 patients presented with perineal and pelvic pain. Other symptoms such as fever, urine retention, and recurrent UTI were variably reported by others. Table 1 summarizes the clinical presentation and management of bladder perforation in the previously published case reports. Table1. A summary of the presentation and management of bladder perforation after immediate chemotheraputic agent intravesical instillation. Ref. number The instilled drug Number of patients Initial presentation Management outline Lim et al7 MMC 1 Pelvic and suprapubic pain Exploratry laparotomy and bladder repair after failed conservative management Elmamoun et al9 MMC 6 3 patients presented with pelvic pain Conservative management and urethral catheterization for 2, 26, and 52 weeks Pelvic pain Recurrent UTIs Left ureteric obstruction Partial cystectomy + left to right trans-ureteroureterostomy Pelvic pain, Persistent urinary leak Left ureteric obstruction Augmentation cystoplasty + leftUreteric reimplantationThe patient ended with persistent leak, so a long-term catheter was used Pelvic painSevere LUTS Bilateral ureteric obstruction Urinary diversion (ileal conduit) Penna et al10 MMC 1 Lower abdominal pain and inability to void Analgesia, antibiotics and urethral catheter for 4 weeks Tyritzis et al11 EPI 2 Fever and retropubic pain Conservative: Antibiotics and urethral catheter Fever, lower abdominal pain and cellulitis Exploratry laparotomy and bladder repair after failed conservative management Nieuwenhuijzen et al12 MMC Pelvic pain and fever Exploratry laparotomy and bladder repair after failed conservative management for 1 month Racioppi et al13 MMC Pelvic pain, fever and pancytopenia Exploratry laparotomy and bladder repair after failed conservative management for 3 weeks Hatem and Leifeld14 MMC Pelvic pain Fever and positive blood culture for ecoli as well as purulent discharge from the drain site after the first expolratory laparatomy Conservative management for 10 daysExploratory laparatomy that revealed edematous anterior bladder wall without defectRenewed laparotomy, drainage of the abscess, debridement of the necrotic tissue, mono-J ureteral stents and nephrostomyRadical cystectomy with a urinary diversion through ureterocutaneostomy Shenaq et al15 EPI 3 Strong urge and pain in the lower left abdomen Conservative management with antibiotics and urethral catheterization Abdominal pain and intestinal obstruction Conservative management for 12 daysExploratory laparotomy that revealed infiltrative mass on the top of the bladder causing IO, so colostomy was done.The patient died due to multi organ failure Progressive pain in the lower abdomen Conservative management with antibiotics and urethral catheterization Abbreviations: Ep, epirubicin; MMC, mitomycin C. Concerning the management, almost all the reported cases were initially managed conservatively by urethral catheterization, anticholinergics, and antibiotics. However, this was insufficient to heal the bladder in some cases which needed open repair. Moreover, some ended with cystectomy. In our case, the patient has an extraperitoneal extravasation that failed the conservative management and needed open surgical repair. A striking feature of the healing process that was seen in many cases including ours is the poor and slow healing. In our case, the patient needed 9 weeks for complete healing after proper open 3 layers bladder wall repair.7-9,13,14 While Lim et al attributed the slow healing in their case to the patient’s pre-existing peripheral vascular disease and suboptimal tissue oxygenation, no risk factors were identified in the other cases including our patient who was free of any comorbidities. We suppose that the poor healing is linked to the chemotherapeutic agent inflammatory effect rather than to patient factor. This assumption is backed by the reported characteristic of mitomycin induced skin ulcer in cases of extravasation after intravenous administration. These ulcers are well known for their chronicity and the low tendency of spontaneous healing. Moreover, debridement and soft-tissue reconstruction following extravasation of MMC are recommended for patients with persistent ulceration and pain.15 Another common finding in the cases that mandate open repair was the presence of perivesical soft tissue necrosis and extensive inflammation. Doherty et al observed that more extensive bladder wall and fat necrosis of extravesical tissue in cystectomy specimens occurred when either epirubicin or mitomycin instillation was administered than in that seen following TURBT alone.16 This cytotoxic effect was also reported by Tyritzis et al11 after epirubicin instillation, and by Cliff et al,8 Nieuwenhuijzen et al,12 and Hatem and Leifeld14 after MMC instillation. This cytotoxic effect was recognized herein for the doxorubicin instillation, which is the first time to be reported as a complication of doxorubicin. It is worth to mention that the perforation had no adverse oncological effect on our patient as no tumor recurrence was detected during follow up. Conclusion Immediate intravesical instillations of chemotherapy are not devoid from risk and serious complications as bladder perforation can occur. Whether it’s value in decreasing the recurrence justify the risk of bladder perforation is not our argument in this case report. Nevertheless, this severe complication should be kept in mind, and high index of suspicion will aid in timely management. If perforation is suspected, urethral catheterization and contrasted imaging are mandatory. In the case of failed conservative management surgical repair is warranted even in extraperitoneal leak. Funding:The author(s) received no financial support for the research, authorship, and/or publication of this article. Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Author Contributions: Concept: AA, SAD. Design: MA, SA. Supervision: AA, SAD, MAA. Resources: MA, HM, NB. Materials: AA, MA, NB. Writing manuscript: AA, MA, HM. Critical review: AA, SAD, MAA. Informed Consent: The patient has given his written informed consent to publish his case including publication of images. The study follows the guidelines for human studies and is conducted in accordance with the World Medical Association Declaration of Helsinki. ORCID iD: Hammam Mansi https://orcid.org/0000-0002-4571-4336	DOXORUBICIN	DrugsGivenReaction	CC BY-NC	33473244	18,858,074	2021
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Product use issue'.	Delayed Bladder Perforation Related to Immediate Single Dose Intravesical Doxorubicin Instillation After TURBT: A Case Report and Literature Review. Intravesical chemotherapy instillation immediately after tumor resection is a well-known practice in the management of non-muscle invasive bladder cancer. Despite being largely well tolerated in most cases, it is not devoid of severe and life-threatening complications. We present an unusual case of bladder perforation that happened 2 weeks after bladder tumor resection. The patient had received single dose intra-vesical instillation of doxorubicin after TUR-BT. Conservative managements failed to achieve bladder healing; as a result, open surgical repair was performed. To the best of our knowledge, this is the first reported case of bladder perforation after intra-vesical doxorubicin instillation. The occurrence of such a rare serious complication in a mostly safe intervention must be taken into consideration. A high index of suspicion, timely management, and proceeding to more invasive surgical treatments when necessary are cornerstones in the management and preserving the bladder. Introduction Bladder cancer (BC) is the seventh most commonly diagnosed cancer in men worldwide. About 75% of the cases are Non-muscle invasive (NMIBC).1 Transurethral resection of the bladder tumor (TURBT) remains the gold standard of therapy for diagnosing and treating NMIBC. To decrease the recurrence rate of low-risk tumors, a single, immediate, post-operative intravesical instillation of chemotherapy is recommended.1 The most commonly used chemotherapeutic agent is Mitomycin C (MMC), which is an alkylating agent that inhibits DNA synthesis and causes single-strand breakage of DNA and chromosomal breaks.2 Immediate single instillation (SI) is largely well-tolerated. However, severe complications and even death have been reported in the literature. Here we present a case wherein the patient had an extraperitoneal bladder perforation that occurred 2 weeks after TUT-BT and immediate postoperative instillation of doxorubicin. Case Presentation A 63-Year-old male, a heavy smoker, with no other co-morbidities, was referred to urology clinic for evaluation and management of a lesion on the left bladder wall that was incidentally detected on a CT scan. He did not have any urinary symptoms on initial presentation. Cystourethroscopy revealed 2 papillary growths on the left lateral bladder wall, which were resected completely and smoothly by a resectoscope using a monopolar energy. Complete resection of the tumor was achieved without any intra-operative complication or perforation. Hemostasis was secured, and a urethral catheter was inserted. In accordance of the EAU guidelines, the patient received an intravesical instillation of 50 ml of doxorubicin diluted in 100 cc of saline 6 hours after the TURBT. During the instillation, his vital signs were stable and he had neither hematuria nor abdominal pain. One hour after the instillation, the catheter was unclamped and was attached to continuous bladder irrigation. Two days after the surgery and in the absence of hematuria, the indwelling catheter was removed, and the patient was discharged home the next day. When seen in the clinic 1 week later the patient was fine with no alarming symptoms. The pathology report showed a low-grade Ta Papillary urothelial carcinoma. Based on the pathological diagnosis and according to the EAU guidelines, adjuvant chemotherapy instillations were planned. Two weeks later, the patient presented with, dysuria, frequency, urgency, as well as pelvic and perineal pain. Physical examination was unremarkable. Urine analysis showed numerous WBC’s, RBC’s, and bacteria. An empirical antibiotics course was commenced. In view of the worsening symptoms and the past history of anal fistula, a contrast enhanced pelvic MRI was performed. It showed a diffusely thickened urinary bladder wall, with evidence of a focal wall defect at its left aspect (the site of the resected tumor). The active urine leak into the perivesical space confirmed the diagnosis of retroperitoneal bladder perforation (Figure 1). Figure. 1. Pelvic MRI (axial view) showing extraperitoneal bladder perforation at the left lateral wall (arrow points to extravasated contrast). The patient was managed conservatively with antibiotics and an indwelling catheter for 3 weeks. A follow-up retrograde CT cystogram showed no improvement and a gross contrast leak from the same site was identified. The catheter was kept for 6 more weeks and the CT cystogram was repeated showing the same results with no evidence of improvement. The patient refused to keep the catheter for a longer period so the option of open surgical repair was discussed with him. Extraperitoneal laparotomy was performed, a perivesical small abscess was drained and cultured, necrotic tissues were debrided, and the bladder wall defect was rimed and closed in 3 layers. Tissue biopsies from the pelvic wall and bladder wall were harvested. A urethral catheter and pelvic drain were kept in place. The postoperative course was uneventful, the drain was removed on the third-day postoperatively, and the patient was discharged home with the urethral catheter in. Histopathological evaluation of the pelvic wall and the bladder biopsies verified acute and chronic inflammation as well as granulation tissue formation. However, both were negative for malignancy. The urinary bladder biopsy also showed extensive foci of dystrophic calcification while the pelvic wall biopsy revealed fat necrosis (Figures 2 and 3). Figure 2. The histologic examination revealed fragments of soft tissue with fat necrosis in the lower left area, mixed acute and chronic inflammatory cell infiltrate and hemorrhage in the upper right area (Hematoxylin & Eosin, 200X). Figure 3. The histologic examination revealed multiple areas of calcifications embedded deep in the tissue with associated crystal-like material suggestive of calcium-oxalate or talc powder crystals (Hematoxylin & Eosin, 200X). A follow up CT cystogram was done 3 weeks after the bladder repair. Although it showed a considerable regression in the size of the bladder defect, interestingly it also showed persistent extra-peritoneal contrast leak from the bladder along it’s left lateral aspect (Figure 4). Figure 4. Retrograde Ct cystogram showing persistent contrast leak from the left lateral aspect after surgical repair. Due to this slow healing, the catheter was kept in. Six weeks later, a follow up CT cystogram showed healing with no extraperitoneal contrast leak (Figure 5). And hence the catheter was removed. Figure 5. Retrograde Ct cystogram showing a healed bladder wall with no contrast leak. Two follow up cystoscopies and bladder biopsy (from an erythematous area on the left lateral wall) with 3 months intervals showed good healing of the bladder wall and no recurrence of the tumor. Discussion Single intravesical instillation with mitomycin C (MMC), epirubicin, or pirarubicin, have shown a beneficial effect in reducing recurrence rate compared to TURBT alone. However, randomized comparisons of individual drugs have not been conducted.3 The high recurrence rate of NMIBC may be attributed to the seeding of floating tumor cells. Immediate chemotherapy instillation has been shown to act by destroying these circulating tumor cells after TURBT. Hence the EAU guidelines recommend initiating the instillation within the first few hours after TURBT.1 Most of the complications of intravesical chemotherapy instillation are minor and can be managed medically. Dysuria and urinary frequency and urgency were the most common complications.4 On the other hand, reporting major complications such as bladder perforation, perirectal abscesses, eosinophilic cystitis, and ureteral stenosis is limited to case reports.5-14 In this case, we described our experience with bladder perforation after immediate intravesical instillation of doxorubicin. One can argue that the perforation has occurred intraoperatively and gone unnoticed. However, in view of the absence of endoscopic evidence of perforation, late presentation of symptoms, and the absence of symptoms during and after the instillation we assume that the Single Dose Intravesical Doxorubicin Instillation caused the perforation rather than complicating an existing one. To explain the mechanism of perforation, Lim et al7 and Cliff et al8 hypothesized that perforation is caused when attenuated muscularis propria gets necrosed after MMC instillation. The presenting complaints of our patient were perineal and pelvic pain as well as urinary irritative symptoms. This is similar to Elmamoun et al9 cohort in which all the 6 patients presented with perineal and pelvic pain. Other symptoms such as fever, urine retention, and recurrent UTI were variably reported by others. Table 1 summarizes the clinical presentation and management of bladder perforation in the previously published case reports. Table1. A summary of the presentation and management of bladder perforation after immediate chemotheraputic agent intravesical instillation. Ref. number The instilled drug Number of patients Initial presentation Management outline Lim et al7 MMC 1 Pelvic and suprapubic pain Exploratry laparotomy and bladder repair after failed conservative management Elmamoun et al9 MMC 6 3 patients presented with pelvic pain Conservative management and urethral catheterization for 2, 26, and 52 weeks Pelvic pain Recurrent UTIs Left ureteric obstruction Partial cystectomy + left to right trans-ureteroureterostomy Pelvic pain, Persistent urinary leak Left ureteric obstruction Augmentation cystoplasty + leftUreteric reimplantationThe patient ended with persistent leak, so a long-term catheter was used Pelvic painSevere LUTS Bilateral ureteric obstruction Urinary diversion (ileal conduit) Penna et al10 MMC 1 Lower abdominal pain and inability to void Analgesia, antibiotics and urethral catheter for 4 weeks Tyritzis et al11 EPI 2 Fever and retropubic pain Conservative: Antibiotics and urethral catheter Fever, lower abdominal pain and cellulitis Exploratry laparotomy and bladder repair after failed conservative management Nieuwenhuijzen et al12 MMC Pelvic pain and fever Exploratry laparotomy and bladder repair after failed conservative management for 1 month Racioppi et al13 MMC Pelvic pain, fever and pancytopenia Exploratry laparotomy and bladder repair after failed conservative management for 3 weeks Hatem and Leifeld14 MMC Pelvic pain Fever and positive blood culture for ecoli as well as purulent discharge from the drain site after the first expolratory laparatomy Conservative management for 10 daysExploratory laparatomy that revealed edematous anterior bladder wall without defectRenewed laparotomy, drainage of the abscess, debridement of the necrotic tissue, mono-J ureteral stents and nephrostomyRadical cystectomy with a urinary diversion through ureterocutaneostomy Shenaq et al15 EPI 3 Strong urge and pain in the lower left abdomen Conservative management with antibiotics and urethral catheterization Abdominal pain and intestinal obstruction Conservative management for 12 daysExploratory laparotomy that revealed infiltrative mass on the top of the bladder causing IO, so colostomy was done.The patient died due to multi organ failure Progressive pain in the lower abdomen Conservative management with antibiotics and urethral catheterization Abbreviations: Ep, epirubicin; MMC, mitomycin C. Concerning the management, almost all the reported cases were initially managed conservatively by urethral catheterization, anticholinergics, and antibiotics. However, this was insufficient to heal the bladder in some cases which needed open repair. Moreover, some ended with cystectomy. In our case, the patient has an extraperitoneal extravasation that failed the conservative management and needed open surgical repair. A striking feature of the healing process that was seen in many cases including ours is the poor and slow healing. In our case, the patient needed 9 weeks for complete healing after proper open 3 layers bladder wall repair.7-9,13,14 While Lim et al attributed the slow healing in their case to the patient’s pre-existing peripheral vascular disease and suboptimal tissue oxygenation, no risk factors were identified in the other cases including our patient who was free of any comorbidities. We suppose that the poor healing is linked to the chemotherapeutic agent inflammatory effect rather than to patient factor. This assumption is backed by the reported characteristic of mitomycin induced skin ulcer in cases of extravasation after intravenous administration. These ulcers are well known for their chronicity and the low tendency of spontaneous healing. Moreover, debridement and soft-tissue reconstruction following extravasation of MMC are recommended for patients with persistent ulceration and pain.15 Another common finding in the cases that mandate open repair was the presence of perivesical soft tissue necrosis and extensive inflammation. Doherty et al observed that more extensive bladder wall and fat necrosis of extravesical tissue in cystectomy specimens occurred when either epirubicin or mitomycin instillation was administered than in that seen following TURBT alone.16 This cytotoxic effect was also reported by Tyritzis et al11 after epirubicin instillation, and by Cliff et al,8 Nieuwenhuijzen et al,12 and Hatem and Leifeld14 after MMC instillation. This cytotoxic effect was recognized herein for the doxorubicin instillation, which is the first time to be reported as a complication of doxorubicin. It is worth to mention that the perforation had no adverse oncological effect on our patient as no tumor recurrence was detected during follow up. Conclusion Immediate intravesical instillations of chemotherapy are not devoid from risk and serious complications as bladder perforation can occur. Whether it’s value in decreasing the recurrence justify the risk of bladder perforation is not our argument in this case report. Nevertheless, this severe complication should be kept in mind, and high index of suspicion will aid in timely management. If perforation is suspected, urethral catheterization and contrasted imaging are mandatory. In the case of failed conservative management surgical repair is warranted even in extraperitoneal leak. Funding:The author(s) received no financial support for the research, authorship, and/or publication of this article. Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Author Contributions: Concept: AA, SAD. Design: MA, SA. Supervision: AA, SAD, MAA. Resources: MA, HM, NB. Materials: AA, MA, NB. Writing manuscript: AA, MA, HM. Critical review: AA, SAD, MAA. Informed Consent: The patient has given his written informed consent to publish his case including publication of images. The study follows the guidelines for human studies and is conducted in accordance with the World Medical Association Declaration of Helsinki. ORCID iD: Hammam Mansi https://orcid.org/0000-0002-4571-4336	DOXORUBICIN HYDROCHLORIDE	DrugsGivenReaction	CC BY-NC	33473244	18,802,244	2021
What was the administration route of drug 'DOXORUBICIN HYDROCHLORIDE'?	Delayed Bladder Perforation Related to Immediate Single Dose Intravesical Doxorubicin Instillation After TURBT: A Case Report and Literature Review. Intravesical chemotherapy instillation immediately after tumor resection is a well-known practice in the management of non-muscle invasive bladder cancer. Despite being largely well tolerated in most cases, it is not devoid of severe and life-threatening complications. We present an unusual case of bladder perforation that happened 2 weeks after bladder tumor resection. The patient had received single dose intra-vesical instillation of doxorubicin after TUR-BT. Conservative managements failed to achieve bladder healing; as a result, open surgical repair was performed. To the best of our knowledge, this is the first reported case of bladder perforation after intra-vesical doxorubicin instillation. The occurrence of such a rare serious complication in a mostly safe intervention must be taken into consideration. A high index of suspicion, timely management, and proceeding to more invasive surgical treatments when necessary are cornerstones in the management and preserving the bladder. Introduction Bladder cancer (BC) is the seventh most commonly diagnosed cancer in men worldwide. About 75% of the cases are Non-muscle invasive (NMIBC).1 Transurethral resection of the bladder tumor (TURBT) remains the gold standard of therapy for diagnosing and treating NMIBC. To decrease the recurrence rate of low-risk tumors, a single, immediate, post-operative intravesical instillation of chemotherapy is recommended.1 The most commonly used chemotherapeutic agent is Mitomycin C (MMC), which is an alkylating agent that inhibits DNA synthesis and causes single-strand breakage of DNA and chromosomal breaks.2 Immediate single instillation (SI) is largely well-tolerated. However, severe complications and even death have been reported in the literature. Here we present a case wherein the patient had an extraperitoneal bladder perforation that occurred 2 weeks after TUT-BT and immediate postoperative instillation of doxorubicin. Case Presentation A 63-Year-old male, a heavy smoker, with no other co-morbidities, was referred to urology clinic for evaluation and management of a lesion on the left bladder wall that was incidentally detected on a CT scan. He did not have any urinary symptoms on initial presentation. Cystourethroscopy revealed 2 papillary growths on the left lateral bladder wall, which were resected completely and smoothly by a resectoscope using a monopolar energy. Complete resection of the tumor was achieved without any intra-operative complication or perforation. Hemostasis was secured, and a urethral catheter was inserted. In accordance of the EAU guidelines, the patient received an intravesical instillation of 50 ml of doxorubicin diluted in 100 cc of saline 6 hours after the TURBT. During the instillation, his vital signs were stable and he had neither hematuria nor abdominal pain. One hour after the instillation, the catheter was unclamped and was attached to continuous bladder irrigation. Two days after the surgery and in the absence of hematuria, the indwelling catheter was removed, and the patient was discharged home the next day. When seen in the clinic 1 week later the patient was fine with no alarming symptoms. The pathology report showed a low-grade Ta Papillary urothelial carcinoma. Based on the pathological diagnosis and according to the EAU guidelines, adjuvant chemotherapy instillations were planned. Two weeks later, the patient presented with, dysuria, frequency, urgency, as well as pelvic and perineal pain. Physical examination was unremarkable. Urine analysis showed numerous WBC’s, RBC’s, and bacteria. An empirical antibiotics course was commenced. In view of the worsening symptoms and the past history of anal fistula, a contrast enhanced pelvic MRI was performed. It showed a diffusely thickened urinary bladder wall, with evidence of a focal wall defect at its left aspect (the site of the resected tumor). The active urine leak into the perivesical space confirmed the diagnosis of retroperitoneal bladder perforation (Figure 1). Figure. 1. Pelvic MRI (axial view) showing extraperitoneal bladder perforation at the left lateral wall (arrow points to extravasated contrast). The patient was managed conservatively with antibiotics and an indwelling catheter for 3 weeks. A follow-up retrograde CT cystogram showed no improvement and a gross contrast leak from the same site was identified. The catheter was kept for 6 more weeks and the CT cystogram was repeated showing the same results with no evidence of improvement. The patient refused to keep the catheter for a longer period so the option of open surgical repair was discussed with him. Extraperitoneal laparotomy was performed, a perivesical small abscess was drained and cultured, necrotic tissues were debrided, and the bladder wall defect was rimed and closed in 3 layers. Tissue biopsies from the pelvic wall and bladder wall were harvested. A urethral catheter and pelvic drain were kept in place. The postoperative course was uneventful, the drain was removed on the third-day postoperatively, and the patient was discharged home with the urethral catheter in. Histopathological evaluation of the pelvic wall and the bladder biopsies verified acute and chronic inflammation as well as granulation tissue formation. However, both were negative for malignancy. The urinary bladder biopsy also showed extensive foci of dystrophic calcification while the pelvic wall biopsy revealed fat necrosis (Figures 2 and 3). Figure 2. The histologic examination revealed fragments of soft tissue with fat necrosis in the lower left area, mixed acute and chronic inflammatory cell infiltrate and hemorrhage in the upper right area (Hematoxylin & Eosin, 200X). Figure 3. The histologic examination revealed multiple areas of calcifications embedded deep in the tissue with associated crystal-like material suggestive of calcium-oxalate or talc powder crystals (Hematoxylin & Eosin, 200X). A follow up CT cystogram was done 3 weeks after the bladder repair. Although it showed a considerable regression in the size of the bladder defect, interestingly it also showed persistent extra-peritoneal contrast leak from the bladder along it’s left lateral aspect (Figure 4). Figure 4. Retrograde Ct cystogram showing persistent contrast leak from the left lateral aspect after surgical repair. Due to this slow healing, the catheter was kept in. Six weeks later, a follow up CT cystogram showed healing with no extraperitoneal contrast leak (Figure 5). And hence the catheter was removed. Figure 5. Retrograde Ct cystogram showing a healed bladder wall with no contrast leak. Two follow up cystoscopies and bladder biopsy (from an erythematous area on the left lateral wall) with 3 months intervals showed good healing of the bladder wall and no recurrence of the tumor. Discussion Single intravesical instillation with mitomycin C (MMC), epirubicin, or pirarubicin, have shown a beneficial effect in reducing recurrence rate compared to TURBT alone. However, randomized comparisons of individual drugs have not been conducted.3 The high recurrence rate of NMIBC may be attributed to the seeding of floating tumor cells. Immediate chemotherapy instillation has been shown to act by destroying these circulating tumor cells after TURBT. Hence the EAU guidelines recommend initiating the instillation within the first few hours after TURBT.1 Most of the complications of intravesical chemotherapy instillation are minor and can be managed medically. Dysuria and urinary frequency and urgency were the most common complications.4 On the other hand, reporting major complications such as bladder perforation, perirectal abscesses, eosinophilic cystitis, and ureteral stenosis is limited to case reports.5-14 In this case, we described our experience with bladder perforation after immediate intravesical instillation of doxorubicin. One can argue that the perforation has occurred intraoperatively and gone unnoticed. However, in view of the absence of endoscopic evidence of perforation, late presentation of symptoms, and the absence of symptoms during and after the instillation we assume that the Single Dose Intravesical Doxorubicin Instillation caused the perforation rather than complicating an existing one. To explain the mechanism of perforation, Lim et al7 and Cliff et al8 hypothesized that perforation is caused when attenuated muscularis propria gets necrosed after MMC instillation. The presenting complaints of our patient were perineal and pelvic pain as well as urinary irritative symptoms. This is similar to Elmamoun et al9 cohort in which all the 6 patients presented with perineal and pelvic pain. Other symptoms such as fever, urine retention, and recurrent UTI were variably reported by others. Table 1 summarizes the clinical presentation and management of bladder perforation in the previously published case reports. Table1. A summary of the presentation and management of bladder perforation after immediate chemotheraputic agent intravesical instillation. Ref. number The instilled drug Number of patients Initial presentation Management outline Lim et al7 MMC 1 Pelvic and suprapubic pain Exploratry laparotomy and bladder repair after failed conservative management Elmamoun et al9 MMC 6 3 patients presented with pelvic pain Conservative management and urethral catheterization for 2, 26, and 52 weeks Pelvic pain Recurrent UTIs Left ureteric obstruction Partial cystectomy + left to right trans-ureteroureterostomy Pelvic pain, Persistent urinary leak Left ureteric obstruction Augmentation cystoplasty + leftUreteric reimplantationThe patient ended with persistent leak, so a long-term catheter was used Pelvic painSevere LUTS Bilateral ureteric obstruction Urinary diversion (ileal conduit) Penna et al10 MMC 1 Lower abdominal pain and inability to void Analgesia, antibiotics and urethral catheter for 4 weeks Tyritzis et al11 EPI 2 Fever and retropubic pain Conservative: Antibiotics and urethral catheter Fever, lower abdominal pain and cellulitis Exploratry laparotomy and bladder repair after failed conservative management Nieuwenhuijzen et al12 MMC Pelvic pain and fever Exploratry laparotomy and bladder repair after failed conservative management for 1 month Racioppi et al13 MMC Pelvic pain, fever and pancytopenia Exploratry laparotomy and bladder repair after failed conservative management for 3 weeks Hatem and Leifeld14 MMC Pelvic pain Fever and positive blood culture for ecoli as well as purulent discharge from the drain site after the first expolratory laparatomy Conservative management for 10 daysExploratory laparatomy that revealed edematous anterior bladder wall without defectRenewed laparotomy, drainage of the abscess, debridement of the necrotic tissue, mono-J ureteral stents and nephrostomyRadical cystectomy with a urinary diversion through ureterocutaneostomy Shenaq et al15 EPI 3 Strong urge and pain in the lower left abdomen Conservative management with antibiotics and urethral catheterization Abdominal pain and intestinal obstruction Conservative management for 12 daysExploratory laparotomy that revealed infiltrative mass on the top of the bladder causing IO, so colostomy was done.The patient died due to multi organ failure Progressive pain in the lower abdomen Conservative management with antibiotics and urethral catheterization Abbreviations: Ep, epirubicin; MMC, mitomycin C. Concerning the management, almost all the reported cases were initially managed conservatively by urethral catheterization, anticholinergics, and antibiotics. However, this was insufficient to heal the bladder in some cases which needed open repair. Moreover, some ended with cystectomy. In our case, the patient has an extraperitoneal extravasation that failed the conservative management and needed open surgical repair. A striking feature of the healing process that was seen in many cases including ours is the poor and slow healing. In our case, the patient needed 9 weeks for complete healing after proper open 3 layers bladder wall repair.7-9,13,14 While Lim et al attributed the slow healing in their case to the patient’s pre-existing peripheral vascular disease and suboptimal tissue oxygenation, no risk factors were identified in the other cases including our patient who was free of any comorbidities. We suppose that the poor healing is linked to the chemotherapeutic agent inflammatory effect rather than to patient factor. This assumption is backed by the reported characteristic of mitomycin induced skin ulcer in cases of extravasation after intravenous administration. These ulcers are well known for their chronicity and the low tendency of spontaneous healing. Moreover, debridement and soft-tissue reconstruction following extravasation of MMC are recommended for patients with persistent ulceration and pain.15 Another common finding in the cases that mandate open repair was the presence of perivesical soft tissue necrosis and extensive inflammation. Doherty et al observed that more extensive bladder wall and fat necrosis of extravesical tissue in cystectomy specimens occurred when either epirubicin or mitomycin instillation was administered than in that seen following TURBT alone.16 This cytotoxic effect was also reported by Tyritzis et al11 after epirubicin instillation, and by Cliff et al,8 Nieuwenhuijzen et al,12 and Hatem and Leifeld14 after MMC instillation. This cytotoxic effect was recognized herein for the doxorubicin instillation, which is the first time to be reported as a complication of doxorubicin. It is worth to mention that the perforation had no adverse oncological effect on our patient as no tumor recurrence was detected during follow up. Conclusion Immediate intravesical instillations of chemotherapy are not devoid from risk and serious complications as bladder perforation can occur. Whether it’s value in decreasing the recurrence justify the risk of bladder perforation is not our argument in this case report. Nevertheless, this severe complication should be kept in mind, and high index of suspicion will aid in timely management. If perforation is suspected, urethral catheterization and contrasted imaging are mandatory. In the case of failed conservative management surgical repair is warranted even in extraperitoneal leak. Funding:The author(s) received no financial support for the research, authorship, and/or publication of this article. Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Author Contributions: Concept: AA, SAD. Design: MA, SA. Supervision: AA, SAD, MAA. Resources: MA, HM, NB. Materials: AA, MA, NB. Writing manuscript: AA, MA, HM. Critical review: AA, SAD, MAA. Informed Consent: The patient has given his written informed consent to publish his case including publication of images. The study follows the guidelines for human studies and is conducted in accordance with the World Medical Association Declaration of Helsinki. ORCID iD: Hammam Mansi https://orcid.org/0000-0002-4571-4336	Intravesical	DrugAdministrationRoute	CC BY-NC	33473244	18,802,244	2021

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